Patent application title: Rice promoters
Yves Hatzfeld (Lille, FR)
Willem Broekaert (Dilbeek, BE)
IPC8 Class: AC12N1582FI
Class name: Plant, seedling, plant seed, or plant part, per se higher plant, seedling, plant seed, or plant part (i.e., angiosperms or gymnosperms) squash (e.g., pumpkin, zucchini, etc.)
Publication date: 2009-11-12
Patent application number: 20090282587
The invention provides several promoters isolated from Oryza sativa, which
promoters are capable of driving and/or regulating the expression of an
operably linked nucleic acid in a plant. The expression patterns of the
promoters according to the invention have been studied in Oryza sativa
and some of the promoters displayed specific activity in particular
cells, tissues or organs of the plant, while others displayed
constitutive expression throughout substantially the whole plant. Some
promoters showed weak expression, while others were strongly active.
19. An isolated promoter capable of driving and/or regulating expression, comprising: (a) an isolated nucleic acid as given in any one of SEQ ID NO 1 to 22 or the complement of any one of SEQ ID NO 1 to 22; or (b) an isolated nucleic acid having at least 90% sequence identity with any of the DNA sequences as given in any one of SEQ ID NO 1 to 22; or (c) an isolated nucleic acid specifically hybridizing under stringent conditions with any of the DNA sequences as given in any one of SEQ ID NO 1 to 22; or (d) an isolated nucleic acid as defined in any one of (a) to (c), which is interrupted by an intervening sequence; or (e) a fragment of any of the nucleic acids as defined in (a) to (d), which fragment is capable of driving and/or regulating expression.
20. The promoter according to claim 19, which is a hybrid promoter comprising at least one part of a promoter as defined in claim 19 and further comprising another part of a promoter.
21. A genetic construct comprising: (a) an isolated promoter as defined in claim 19; and (b) a heterologous nucleic acid sequence operably linked to said promoter of (a); and optionally (c) a 3' transcription terminator.
22. An expression cassette comprising a genetic construct as defined in claim 21.
23. A transformation vector comprising a genetic construct as defined in claim 21.
24. An expression vector comprising a genetic construct as defined in claim 21.
25. A host cell comprising an isolated promoter as defined in claim 19.
26. A genetic construct as defined in claim 21.
27. An expression cassette as defined in claim 22.
28. A transformation vector as defined in claim 23.
29. An expression vector as defined in claim 24.
30. The host cell according to claim 25, selected from the group consisting of a bacteria, algae, fungi, yeast, plant, insect and animal host cell.
31. A transgenic plant cell comprising an isolated promoter as defined in claim 19.
32. A genetic construct as defined in claim 21.
33. An expression cassette as defined in claim 22.
34. A transformation vector as defined in claim 23.
35. An expression vector as defined in claim 24.
36. The transgenic plant cell according to claim 31, which is a monocot plant cell.
37. The transgenic plant cell according to claim 31, which is a dicot plant cell.
38. A transgenic plant comprising a transgenic plant cell as defined in claim 31.
39. The transgenic plant according to claim 38, wherein said plant is selected from the group consisting of rice, maize, wheat, barley, millet, oats, rye, sorghum, soybean, sunflower, canola, sugarcane, alfalfa, bean, pea, flax, lupinus, rapeseed, tobacco, tomato, potato, squash, papaya, poplar and cotton.
40. A plant part as defined in claim 39.
41. The plant part as defined in claim 40, selected from the group consisting of a harvestable part, a propagule and progeny of the plant.
42. A method for driving and/or regulating expression of a nucleic acid in a plant or plant cell, comprising: (a) operably linking said nucleic acid to any one of the isolated nucleic acids as defined in claim 19, and (b) introducing the resultant genetic construct into a plant or plant cell.
43. The method according to claim 42, wherein said expression is constitutive or tissue-specific.
44. A method for the production of a transgenic plant, comprising: (a) introducing into a plant cell an isolated promoter as defined in claim 19, and (b) cultivating said plant cell under conditions promoting plant growth.
45. A method for the production of a transgenic plant, comprising: (a) introducing into a plant cell a genetic construct as defined in claim 21, and (b) cultivating said plant cell under conditions promoting plant growth.
46. A method for the production of a transgenic plant, comprising: (a) introducing into a plant cell an expression cassette as defined in claim 22, and (b) cultivating said plant cell under conditions promoting plant growth.
47. A method for the production of a transgenic plant, comprising: (a) introducing into a plant cell a transformation vector as defined in claim 23, and (b) cultivating said plant cell under conditions promoting plant growth.
The present invention relates to the field of plant molecular
biology, more particularly to nucleic acid sequences useful for driving
and/or regulating expression of an operably linked nucleic acid in
plants. The isolation of these nucleic acid sequences from rice, as well
as their use in driving and/or regulating expression of an operably
linked nucleic acid is disclosed. The present invention therefore
concerns promoters, hybrid promoters, genetic constructs, expression
cassettes, transformation vectors, expression vectors, host cells and
transgenic plants comprising the isolated nucleic acids according to the
present invention. The present invention also concerns methods for
driving and/or regulating expression of a nucleic acid and methods for
the production of transgenic plants.
Gene expression is dependent on initiation of transcription, which is mediated via the transcription initiation complex. Gene expression is also dependent on regulation of transcription, which regulation determines how strong, when or where a gene is expressed. Said regulation of gene expression may be mediated via transcriptional control elements, which are generally embedded in the nucleic acid sequence 5'-flanking or upstream of the expressed gene. This upstream nucleic acid region is often referred to as a "promoter" since it promotes the binding, formation and/or activation of the transcription initiation complex and therefore is capable of driving and/or regulating expression of the 3' downstream nucleic acid sequence.
Genetic engineering of plants with the aim of obtaining a useful plant phenotype, often involves heterologous gene expression, which is generally mediated by a promoter capable of driving and/or regulating expression of an operably linked heterologous nucleic acid. The phenotype of the host plant only depends on the contribution of the heterologous nucleic acid, but also on the contribution of the specific expression pattern of the chosen promoter determining how, where and when that heterologous nucleic acid is expressed. Accordingly, the choice of promoter with a suitable expression pattern is of crucial importance for obtaining the suitable phenotype. A person skilled in the art will need to have available different promoters, to determine the optimal promoter for a particular nucleic acid. For many different host plants, this availability is rather limited and there is therefore a continuing need to provide new promoters with various expression profiles.
The nucleic acids as presented in SEQ ID NO 1 to 22 were isolated from Oryza sativa and have been found to be capable of driving and regulating expression of an operably linked nucleic acid; their expression patterns have also been characterized. Therefore the present invention offers a collection of hitherto unknown isolated nucleic acids, which isolated nucleic acids are useful as promoters.
Accordingly, the present invention provides an isolated promoter capable of driving and/or regulating expression, comprising: (a) an isolated nucleic acid as given in any one of SEQ ID NO 1 to 22 or the complement of any one of SEQ ID NO 1 to 22; or (b) an isolated nucleic acid having at least 90% sequence identity with any of the DNA sequences as given in any one of SEQ ID NO 1 to 22; or (c) an isolated nucleic acid specifically hybridizing under stringent conditions with any of the DNA sequences as given in any one of SEQ ID NO 1 to 22; or (d) an isolated nucleic acid as defined in any one of (a) to (c), which is interrupted by an intervening sequence; or (e) a fragment of any of the nucleic acids as defined in (a) to (d), which fragment is capable of driving and/or regulating expression.
The term "isolated" as used herein means being removed from its original source. Preferably, the "isolated" promoter is free of sequences (such as protein encoding sequences or other sequences at the 3' end) that naturally flank the promoter in the genomic DNA of the organism from which the promoter is derived. Further preferably, the "isolated" promoter is also free of sequences that naturally flank it at the 5' end. Further preferably, the "isolated" promoter may comprise less than about 5 kb, 4 kb, 3 kb, 2 kb, 1.5 kb, 1.2 kb, 1 kb, 0.8 kb, 0.5 kb or 0.1 kb of nucleotide sequences that naturally occur with the promoter in genomic DNA from the organism of which the promoter is derived.
The present invention is not limited to the nucleic acids as presented by SEQ ID NO 1 to 22. A person skilled in the art will recognize that variants or fragments of a nucleic acid may occur, whilst maintaining the same functionality. These variants or fragments may be man made (e.g. by genetic engineering) or may even occur in nature. Therefore the present invention extends to variant nucleic acids and fragments of any of SEQ ID NO 1 to 22, which variants or fragments are useful in the methods of the present invention. Such variants and fragments include: (a) an isolated nucleic acid as given in any one of SEQ ID NO 1 to 22 or the complement of any one of SEQ ID NO 1 to 22; or (b) an isolated nucleic acid having at least 90% sequence identity with any of the DNA sequences as given in any one of SEQ ID NO 1 to 22; or (c) an isolated nucleic acid specifically hybridizing under stringent conditions with any of the DNA sequences as given in any one of SEQ ID NO 1 to 22; or (d) an isolated nucleic acid as defined in any one of (a) to (c), which is interrupted by an intervening sequence; or (e) a fragment of any of the nucleic acids as defined in (a) to (d), which fragment is capable of driving and/or regulating expression.
Suitable variants of any one of SEQ ID NO 1 to 22 encompass homologues which have in increasing order of preference at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with any one of the nucleic acids as represented in SEQ ID NO 1 to 22.
The percentage of identity may be calculated using an alignment program. Preferably a pair wise global alignment program may be used, which implements the algorithm of Needleman-Wunsch (J. Mol. Biol. 48: 443-453, 1970). This algorithm maximizes the number of matches and minimizes the number of gaps. Such programs are for example GAP, Needle (EMBOSS package), stretcher (EMBOSS package) or Align X (Vector NTI suite 5.5) and may use the standard parameters (for example gap opening penalty 15 and gap extension penalty 6.66). Alternatively, a local alignment program implementing the algorithm of Smith-Waterman (Advances in Applied Mathematics 2, 482-489 (1981)) may be used. Such programs are for example Water (EMBOSS package) or matcher (EMBOSS package). "Sequence identity" as used herein is preferably calculated over the entire length of the promoters as represented by any one of SEQ ID NO 1 to 22. The length of these promoters is presented in Table 2.
Search and identification of homologous nucleic acids, would be well within the realm of a person skilled in the art. Such methods involve screening sequence databases with the sequences provided by the present invention, for example any one of SEQ ID NO 1 to 22, preferably in a computer readable form. Useful sequence databases include but are not limited to Genbank, the European Molecular Biology Laboratory Nucleic acid Database (EMBL) or versions thereof, or the MIPS database. Different search algorithms and software for the alignment and comparison of sequences are well known in the art. Such software includes, for example GAP, BESTFIT, BLAST, FASTA and TFASTA. Preferably BLAST software is used, which calculates percent sequence identity and performs a statistical analysis of the similarity between the sequences. The suite of programs referred to as BLAST programs has 5 different implementations: three designed for nucleotide sequence queries (BLASTN, BLASTX, and TBLASTX) and two designed for protein sequence queries (BLASTP and TBLASTN) (Coulson, Trends in Biotechnology: 76-80, 1994; Birren et al., GenomeAnalysis, 1: 543, 1997). The software for performing BLAST analysis is publicly available through the National Centre for Biotechnology Information.
The sequences of the genome of Arabidopsis thaliana and the genome of Oryza sativa are now available in public databases such as Genbank. Other genomes are currently being sequenced. Therefore, it is expected that as more sequences of the genomes of other plants become available, homologous promoters may be identifiable by sequence alignment with any one of SEQ ID NO 1 to SEQ ID NO 22. The skilled person will readily be able to find homologous promoters from other plant species, for example from other crop plants, such as maize. Homologous promoters from other crop plants are especially useful for practising the methods of the present invention in crop plants.
One example of homologues having at least 90% sequence identity with any one of SEQ ID NO to 22 are allelic variants of any one of SEQ ID NO 1 to 22. Allelic variants are variants of the same gene occurring in two different individuals of the same species and usually allelic variants differ by slight sequence changes. Allelic variants may encompass Single Nucleotide Polymorphisms (SNPs) as well as Small Insertion/Deletion Polymorphisms (INDELs). The size of INDELs is usually less than 100 bp. SNPs and INDELs form the largest set of sequence variants in naturally occurring polymorphic strains of most organisms.
Homologues suitable for use in the methods according to the invention may readily be isolated from their source organism via the technique of PCR or hybridization. Their capability of driving and/or regulating expression may readily be determined, for example, by following the methods described in the Examples section by simply substituting the sequence used in the actual Example with the homologue.
Other suitable variants of any one of SEQ ID NO 1 to 22 encompassed by the present invention are nucleic acids specifically hybridising under stringent conditions to any one of the nucleic acids of SEQ ID NO 1 to 22. The term "hybridising" means annealing to substantially homologous complementary nucleotide sequences in a hybridization process. Tools in molecular biology relying on such a hybridization process include the polymerase chain reaction (PCR; and all methods based thereon), subtractive hybridisation, random primer extension, nuclease S1 mapping, primer extension, reverse transcription, cDNA synthesis, differential display of RNAs, and DNA sequence determination, Northern blotting (RNA blotting), Southern blotting (DNA blotting). The hybridisation process can also occur with one of the complementary nucleic acids immobilised to a matrix such as magnetic beads, Sepharose beads or any other resin. Tools in molecular biology relying on such a process include the isolation of poly (A+) mRNA. The hybridisation process can furthermore occur with one of the complementary nucleic acids immobilised to a solid support such as a nitro-cellulose or nylon membrane or immobilised by e.g. photolithography to, for example, a siliceous glass support (the latter known as nucleic acid arrays or microarrays or as nucleic acid chips). Tools in molecular biology relying on such a process include RNA and DNA gel blot analysis, colony hybridisation, plaque hybridisation, in situ hybridisation and microarray hybridisation. In order to allow hybridisation to occur, the nucleic acid molecules are generally thermally or chemically denatured to melt a double strand into two single strands and/or to remove hairpins or other secondary structures from single stranded nucleic acids. The stringency of hybridisation is influenced by conditions such as temperature, salt concentration and hybridisation buffer composition. Conventional hybridisation conditions are described in, for example, Sambrook (2001) Molecular Cloning: a laboratory manual, 3rd Edition Cold Spring Harbor Laboratory Press, CSH, New York, but the skilled craftsman will appreciate that numerous different hybridisation conditions can be designed in function of the known or the expected homology and/or length of the nucleic acid sequence. High stringency conditions for hybridisation include high temperature and/or low sodium/salt concentration (salts include sodium as for example in NaCl and Na3-citrate) and/or the inclusion of formamide in the hybridisation buffer and/or lowering the concentration of compounds such as SDS (sodium dodecyl sulphate detergent) in the hybridisation buffer and/or exclusion of compounds such as dextran sulphate or polyethylene glycol (promoting molecular crowding) from the hybridisation buffer. Specifically hybridising under stringent conditions means that the sequences have to be very similar. Specific hybridization under stringent conditions is preferably carried out at a temperature of 60° C. followed by washes in 0.1 to 1×SSC, 0.1×SDS, and 1×SSC, 0.1×SDS.
The invention also relates to a nucleic acid molecule of at least 15 nucleotides in length hybridizing specifically with any of the nucleic acids of the invention. The invention also relates to a nucleic acid molecule of at least 15 nucleotides in length specifically amplifying a nucleic acid of the invention by polymerase chain reaction.
Another variant of any of SEQ ID NO 1 to 22 encompassed by the present invention are nucleic acids corresponding to any one of SEQ ID NO 1 to 22 or variants thereof as described hereinabove, which are interrupted by an intervening sequence. For example, any of the nucleic acids as presented in SEQ ID NO 1 to 22 may be interrupted by an intervening sequence. With "intervening sequences" is meant any nucleic acid or nucleotide, which disrupts another sequence. Examples of intervening sequences comprise introns, nucleic acid tags, T-DNA and mobilizable nucleic acids sequences such as transposons or nucleic acids that can be mobilized via recombination. Examples of particular transposons comprise Ac (activator), Ds (Dissociation), Spm (suppressor-Mutator) or En. The introduction of introns into promoters is now widely applied. The methods according to the present invention may also be practised using a nucleic acid sequence according to any one of SEQ ID NO 1 to 22 provided with an intron. In case the intervening sequence is an intron, alternative splice variants of the nucleic acids according to the invention may arise. The term "alternative splice variant" as used herein encompasses variants of a nucleic acid sequence in which intervening introns have been excised, replaced or added. Such splice variants may be found in nature or may be manmade. Methods for making such promoters with an intron or for making the corresponding splice variants are well known in the art.
Variants interrupted by an intervening sequence, suitable for use in the methods according to the invention may readily be determined for example by following the methods described in the Examples section by simply substituting the sequence used in the actual Example with the variant.
The variant nucleic acids as described hereinabove may be found in nature (for example allelic variants or splice variants). Additionally and/or alternatively, variants of any one of SEQ ID NO 1 to 22 as described hereinabove may be manmade via techniques well known in the art involving for example mutation, substitution, insertion, deletions or derivation. The present invention also encompasses such variants, as well as their use in the methods of the present invention.
A "mutation variant" of a nucleic acid may readily be made using recombinant DNA manipulation techniques or nucleotide synthesis. Examples of such techniques include site directed mutagenesis via M13 mutagenesis, T7-Gen in vitro mutagenesis (USB, Cleveland, Ohio), QuickChange Site Directed mutagenesis (Stratagene, San Diego, Calif.), PCR-mediated site-directed mutagenesis or other site-directed mutagenesis protocols. Alternatively, the nucleic acid of the present invention may be randomly mutated.
A "substitutional variant" refers to those variants in which at least one residue in the nucleic acid sequence has been removed and a different residue inserted in its place. Nucleic acid substitutions are typically of single residues, but may be clustered depending upon functional constraints placed upon the nucleic acid sequence; insertions usually are of the order of about 1 to about 10 nucleic acid residues, and deletions can range from about 1 to about 20 residues.
An "insertional variant" of a nucleic acid is a variant in which one or more nucleic acid residues are introduced into a predetermined site in that nucleic acid. Insertions may comprise 5'-terminal and/or 3'-terminal fusions as well as intra-sequence insertions of single or multiple nucleotides. Generally, insertions within the nucleic acid sequence will be smaller than 5'- or 3'-terminal fusions, of the order of about 1 to 10 residues. Examples of 5'- or 3'-terminal fusions include the coding sequences of binding domains or activation domains of a transcriptional activator as used in the yeast two-hybrid system or yeast one-hybrid system, or of phage coat proteins, (histidine)6-tag, glutathione S-transferase-tag, protein A, maltose-binding protein, dihydrofolate reductase, Tag•100 epitope, c-myc epitope, FLAG®-epitope, lacZ, CMP (calmodulin-binding peptide), HA epitope, protein C epitope and VSV epitope.
The term "derivative" of a nucleic acid may comprise substitutions, and/or deletions and/or additions of naturally and non-naturally occurring nucleic acid residues compared to the natural nucleic acid. Derivatives may, for example, comprise methylated nucleotides, or artificial nucleotides.
Also encompassed with in the present invention are promoters, comprising a fragment of any of the nucleic acids as presented by any one of SEQ ID NO 1 to 22 or variants thereof as described hereinabove. A "fragment" as used herein means a portion of a nucleic acid sequence. Suitable fragments useful in the methods of the present invention are functional fragments, which retain at least one of the functional parts of the promoter and hence are still capable of driving and/or regulating expression. Examples of functional fragments of a promoter include the minimal promoter, the upstream regulatory elements, or any combination thereof.
Suitable fragments may range from at least about 20 base pairs or about 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950 or 1000 base pairs, up to about the full length sequence of the invention. These base pairs are typically immediately upstream of the transcription initiation start, but alternatively may be from anywhere in the promoter sequence.
Suitable fragments useful in the methods of the present invention may be tested for their capability of driving and/or regulating expression by standard techniques well known to the skilled person, or by the following method described in the Example section.
The promoters as disclosed in any one of SEQ ID NO 1 to 22 are isolated as nucleic acids of approximately 1.2 kb from the upstream region of particular rice coding sequences (CDS). These nucleic acids may include typical elements of a promoter, which are presented in FIG. 1. Generally, a promoter may comprises from coding sequence to the upstream direction: (i) an 5'UTR of pre-messenger RNA, (ii) a minimal promoter comprising the transcription initiation element (INR) and more upstream a TATA box, and (iii) may contain regulatory elements that determine the specific expression pattern of the promoter.
The term "promoter" as used herein is taken in a broad context and refers to regulatory nucleic acid sequences capable of effecting (driving and/or regulating) expression of the sequences to which they are operably linked. A "promoter" encompasses transcriptional regulatory sequences derived from a classical genomic gene. Usually a promoter comprises a TATA box, which is capable of directing the transcription initiation complex to the appropriate transcription initiation start site. However, some promoters do not have a TATA box (TATA-less promoters), but are still fully functional for driving and/or regulating expression. A promoter may additionally comprise a CCAAT box sequence and additional regulatory elements (i.e. upstream activating sequences or cis-elements such as enhancers and silencers). A "promoter" may also include the transcriptional regulatory sequences of a classical prokaryotic gene, in which case it may include a -35 box sequence and/or a -10 box transcriptional regulatory sequences.
"Driving expression" as used herein means promoting the transcription of a nucleic acid.
"Regulating expression" as used herein means influencing the level, time or place of transcription of a nucleic acid. The promoters of the present invention may thus be used to increase, decrease or change in time and/or place transcription of a nucleic acid. For example, they may be used to limit the transcription to certain cell types, tissues or organs, or during a certain period of time, or in response to certain environmental conditions.
The promoter is preferably a plant-expressible promoter. The term "plant-expressible" means being capable of regulating expression in a plant, plant cell, plant tissue and/or plant organ. Accordingly, the invention encompasses an isolated nucleic acid as mentioned above, capable of regulating transcription of an operably linked nucleic acid in a plant or in one or more particular cells, tissues or organs of a plant.
The expression pattern of the promoters according to the present invention were studied in detail and it was found that many of them were tissue-specific. Accordingly, the present invention provides "tissue-specific" promoters. The term "tissue-specific" shall be taken to indicate that expression is predominantly in a particular tissue, tissue-type, organ or any other part of the organism, albeit not necessarily exclusively in said tissue, tissue-type, organ or other part. Accordingly, the invention encompasses an isolated nucleic acid as mentioned above, capable of driving and/or regulating expression (of an operably linked nucleic acid) in a tissue-specific manner. Expression may be driven and/or regulated in the seed, embryo, scutellum, aleurone, endosperm, leaves, flower, calli, meristem, shoot meristem, discriminating centre, shoot, shoot meristem and root. In grasses the shoot meristem is located in the so-called discrimination zone from where the shoot and the leaves originate.
A tissue-specific promoter is one example of a so-called "regulated promoter". These promoters are regulated by endogenous signals such as the presence of certain transcription factors, metabolites, plant hormones, or exogenous signals, such as ageing, stresses or nutritional status. These regulations may have an effect on one or more different levels such spatial specificity or temporal specificity. Encompassed within the present invention is a nucleic acid as described hereinabove, which is a "regulated promoter". Examples of regulated promoters are cell-specific promoters, tissue-specific promoters, organ-specific promoters, cell cycle-specific promoters, inducible promoters or young tissue-specific promoters.
Alternatively and/or additionally, some promoters of the present invention display a constitutive expression pattern. Accordingly, the present invention provides a promoter as described hereinabove, which is a constitutive promoter. The term "constitutive" means having no or very few spatial or temporal regulations. The term "constitutive expression" as used herein refers to a substantially continuously expression in substantially all tissues of the organism. The skilled craftsman will understand that a "constitutive promoter" is a promoter that is active during most, but not necessarily all, phases of growth and development of the organism and throughout most, but not necessarily all, parts of an organism.
The "expression pattern" of a promoter is not only influenced by the spatial and temporal aspects, but also by the level of expression. The level of expression is determined by the so-called "strength" of a promoter. Depending on the resulting expression level, a distinction is made herein between "weak" or "strong" promoters. Generally by "weak promoter" is meant a promoter that drives expression of an operably linked nucleic acid at levels of about 1/10000 transcripts to about 1/100000 transcripts to about 1/500000 transcripts. Generally, by "strong promoter" is meant a promoter that drives expression at levels of about 1/10 transcripts, to about 1/100 or to about 1/1000 transcripts.
According to a particular embodiment, the invention provides an isolated promoter as mentioned hereinabove, which is a hybrid promoter. The term "hybrid promoter" as used herein refers to a chimeric promoter made, for example, synthetically, for example by genetic engineering. Preferred hybrid promoters according to the present invention comprise a part, preferably a functional part, of one of the promoters according to the present invention and at least another part, preferably a functional part of a promoter. The latter part, may be a part of any promoter, including any one of the promoters according to the present invention and other promoters. One example of a hybrid promoter comprises regulatory element(s) of a promoter according to the present invention combined with the minimal promoter of another promoter. Another example of a hybrid promoter is a promoter comprising additional regulatory elements to further enhance its activity and/or to alter its spatial and/or temporal expression pattern.
The present invention also provides use of a functional fragment of any one of SEQ ID NO 1 to 22 or variant thereof for changing the expression pattern of a promoter. In such methods, at least part of any of the nucleic acids according to the present invention are combined with at least one fragment of another promoter.
Further, the invention provides a genetic construct comprising: (a) An isolated promoter as defined hereinabove (b) A heterologous nucleic acid sequence operably linked to isolated promoter of (a), and optionally (c) A 3' transcription terminator
The term "genetic construct" as used herein means a nucleic acid made by genetic engineering.
The term "operably linked" to a promoter as used herein means that the transcription is driven and/or regulated by that promoter. A person skilled in the art will understand that being operably linked to a promoter preferably means that the promoter is positioned upstream (i.e. at the 5'-end) of the operably linked nucleic acid. The distance to the operably linked nucleic acid may be variable, as long as the promoter of the present invention is capable of driving and/or regulating the transcription of the operably linked nucleic acid. For example, between the promoter and the operably linked nucleic acid, there might be a cloning site, an adaptor, a transcription or translation enhancer.
The operably linked nucleic acid may be any coding or non-coding nucleic acid. The operably linked nucleic acid may be in the sense or in the anti-sense direction. Typically in the case of genetic engineering of host cells, the operably linked nucleic acid is to be introduced into the host cell and is intended to change the phenotype of the host cell. Alternatively, the operably linked nucleic acid is an endogenous nucleic acid from the host cell.
The term "heterologous" as used herein is intended to be "heterologous to the promoter of the present invention". A nucleic acid that is heterologous to the promoter of the present invention is not naturally occurring in the nucleic acid sequences flanking the promoter of the present invention when it is in its biological genomic environment. While the nucleic acid may be heterologous to the promoter of the present invention, it may be homologous or native or heterologous or foreign to the plant host cell. The heterologous operably linked nucleic acid may be any nucleic acid (for example encoding any protein), provided that it comprises or it is flanked by at least one nucleotide which is normally not flanking the promoter of the present invention.
The term "transcription terminator" as used in (c) refers to a DNA sequence at the end of a transcriptional unit which signals termination of transcription. Terminators are 3'-non-translated DNA sequences usually containing a polyadenylation signal, which facilitates the addition of polyadenylate sequences to the 3'-end of a primary transcript. Terminators active in and/or isolated from viruses, yeasts, moulds, bacteria, insects, birds, mammals and plants are known and have been described in literature. Examples of terminators suitable for use in the genetic constructs of the present invention include the Agrobacterium tumefaciens nopaline synthase (NOS) gene terminator, the Agrobacterium tumefaciens octopine synthase (OCS) gene terminator sequence, the Cauliflower mosaic virus (CaMV) 35S gene terminator sequence, the Oryza sativa ADP-glucose pyrophosphorylase terminator sequence (t3'Bt2), the Zea mays zein gene terminator sequence, the rbcs-1A gene terminator, and the rbcs-3A gene terminator sequences, amongst others.
The present invention also provides an expression cassette, a transformation vector or a plant expression vector comprising a genetic construct as described above.
An "expression cassette" as meant herein refers to a minimal genetic construct necessary for expression of a nucleic acid. A typical expression cassette comprises a promoter-gene-terminator combination. An expression cassette may additionally comprise cloning sites, for example Gateway® recombination sites or restriction enzyme recognition sites, to allow easy cloning of the operably linked nucleic acid or to allow the easy transfer of the expression cassette into a vector. An expression cassette may further comprise 5' untranslated regions, 3' untranslated regions, a selectable marker, transcription enhancers or translation enhancers.
With "transformation vector" is meant a genetic construct, which may be introduced in an organism by transformation and may be stably maintained in said organism. Some vectors may be maintained in for example Escherichia coli, A. tumefaciens, Saccharomyces cerevisiae or Schizosaccharomyces pombe, while others such as phagemids and cosmid vectors, may be maintained in bacteria and/or viruses. Transformation vectors may be multiplied in their host cell and may be isolated again therefrom to be transformed into another host cell. Vector sequences generally comprise a set of unique sites recognized by restriction enzymes, the multiple cloning site (MCS), wherein one or more non-vector sequence(s) can be inserted. Vector sequences may further comprise an origin of replication which is required for maintenance and/or replication in a specific host cell. Examples of origins of replication include, but are not limited to, the f1-ori and colE1.
"Expression vectors" form a subset of transformation vectors, which, by virtue of comprising the appropriate regulatory sequences, enable expression of the inserted non-vector sequence(s). Expression vectors have been described which are suitable for expression in bacteria (e.g. E. coli), fungi (e.g. S. cerevisiae, S. pombe, Pichia pastoris), insect cells (e.g. baculoviral expression vectors), animal cells (e.g. COS or CHO cells) and plant cells. One suitable expression vector according to the present invention is a plant expression vector, useful for the transformation of plant cells, the stable integration in the plant genome, the maintenance in the plant cell and the expression of the non-vector sequences in the plant cell.
Typically, a plant expression vector according to the present invention comprises a nucleic acid of any one of SEQ ID NO 1 to 22 or a variant thereof as described hereinabove, optionally operably linked to a second nucleic acid. Typically, a plant expressible vector according to the present invention, further comprises T-DNA regions for stable integration into the plant genome (for example the left border and the right border regions of the Ti plasmid).
The genetic constructs of the invention may further comprise a "selectable marker". As used herein, the term "selectable marker" includes any gene, which confers a phenotype to a cell in which it is expressed, to facilitate the identification and/or selection of cells that are transfected or transformed. Suitable markers may be selected from markers that confer antibiotic or herbicide resistance. Cells containing the genetic construct will thus survive antibiotics or herbicide concentrations that kill untransformed cells. Examples of selectable marker genes include genes conferring resistance to antibiotics (such as nptII encoding neomycin phosphotransferase capable of phosphorylating neomycin and kanamycin, or hpt encoding hygromycin phosphotransferase capable of phosphorylating hygromycin), to herbicides (for example bar which provides resistance to Basta; aroA or gox providing resistance against glyphosate), or genes that provide a metabolic trait (such as manA that allows plants to use mannose as sole carbon source). Visual marker genes result in the formation of colour (for example beta-glucuronidase, GUS), luminescence (such as luciferase) or fluorescence (Green Fluorescent Protein, GFP, and derivatives thereof). Further examples of suitable selectable marker genes include the ampicillin resistance (Ampr), tetracycline resistance gene (Tcr), bacterial kanamycin resistance gene (Kanr), phosphinothricin resistance gene, and the chloramphenicol acetyltransferase (CAT) gene, amongst others.
Furthermore, the present invention encompasses a host cell comprising an isolated promoter, or a genetic construct, or an expression cassette, or a transformation vector or an expression vector according to the invention as described hereinabove. In particular embodiments of the invention, the host cell is selected from bacteria, algae, fungi, yeast, plants, insect or animal host cells.
In one particular embodiment, the invention provides a transgenic plant cell comprising an isolated promoter according to the invention, or an isolated nucleic acid, or a genetic construct, or an expression cassette, or a transformation vector or an expression vector according to the invention as described hereinabove. Preferably said plant cell is a dicot plant cell or a monocot plant cell, more preferably a cell of any of the plants as mentioned herein. Preferably, in the transgenic plant cell according to the invention, the promoter or the genetic construct of the invention is stably integrated into the genome of the plant cell.
The invention also provides a method for the production of a transgenic plant, comprising: (a) Introducing into a plant cell an isolated promoter, for example any one of SEQ ID NO 1 to SEQ ID NO 22, or a variant or fragment thereof, or a genetic construct, or an expression cassette, or a transformation vector or an expression vector according to the present invention and as described hereinabove, and (b) Cultivating said plant cell under conditions promoting plant growth.
"Introducing" the above mentioned isolated promoter, or genetic construct, or expression cassette, or transformation vector or expression vector, into a host cell (e.g. plant cell) is preferably achieved by transformation. The term "transformation" as used herein encompasses the transfer of an exogenous polynucleotide into a host cell, irrespective of the method used for transfer. In particular for plants, tissues capable of clonal propagation, whether by organogenesis or embryogenesis, are suitable to be transformed with a genetic construct of the present invention and a whole plant may be regenerated therefrom. The particular tissue chosen will vary depending on the clonal propagation systems available for, and best suited to, the particular plant species being transformed. Exemplary tissue targets include leaf disks, pollen, embryos, cotyledons, hypocotyls, megagametophytes, callus tissue, existing meristematic tissue (e.g., apical meristem, axillary buds, and root meristems), and induced meristem tissue (e.g., cotyledon meristem and hypocotyl meristem). The polynucleotide may be transiently or stably introduced into a plant cell and may be maintained non-integrated, for example, as a plasmid. Alternatively, it may be integrated into the plant genome.
Transformation of a plant species is now a fairly routine technique. Advantageously, any of several transformation methods may be used to introduce the nucleic acids of the invention into a suitable ancestor cell. Transformation methods include the use of liposomes, electroporation, chemicals that increase free DNA uptake, injection of the DNA directly into the plant, particle gun bombardment, transformation using viruses or pollen and microprojection. Methods may be selected from the calcium/polyethylene glycol method for protoplasts (Krens, F. A. et al., 1882, Nature 296, 72-74; Negrutiu I. et al., June 1987, Plant Mol. Biol. 8, 363-373); electroporation of protoplasts (Shillito R. D. et al., 1985 Bio/Technol 3, 1099-1102); microinjection into plant material (Crossway A. et al., 1986, Mol. Gen Genet 202, 179-185); DNA or RNA-coated particle bombardment (Klein T. M. et al., 1987, Nature 327, 70) infection with (non-integrative) viruses and the like. A preferred transformation method for the production of transgenic plant cells according to the present invention, is an Agrobacterium mediated transformation method.
Transgenic rice plants comprising any one of the promoters of the present invention are preferably produced via Agrobacterium-mediated transformation using any of the well-known methods for rice transformation, such as the ones described in any of the following: published European patent application EP 1198985 A1, Aldemita and Hodges (Planta, 199, 612-617, 1996); Chan et al. (Plant Mol. Biol. 22 (3) 491-506, 1993); Hiei et al. (Plant J. 6 (2) 271-282, 1994); which disclosures are incorporated by reference herein as if fully set forth. In the case of corn transformation, the preferred method is as described in either Ishida et al. (Nat. Biotechnol. 1996 June; 14(6): 745-50) or Frame et al. (Plant Physiol. 2002 May; 129(1): 13-22), which disclosures are incorporated by reference herein as if fully set forth.
Generally after transformation, plant cells or cell groupings are selected for the presence of one or more markers which are encoded by plant-expressible genes co-transferred with the gene of interest (which could be under the control of any of the promoters of the present invention), following which the transformed material may be cultivated under conditions promoting plant growth.
The resulting transformed plant cell may then be used to regenerate a transformed plant in a manner known to persons skilled in the art. Accordingly, the method for the production of a transgenic plant as described hereinabove, may further comprise regenerating a plant from said plant cell of (a).
The present invention further provides a plant comprising a plant cell as described hereinabove. The plants may also be able to grow, or even reach maturity including for example fruit production, seed formation, seed ripening and seed setting.
Furthermore, progeny may be produced from these seeds, which progeny may be fertile. Alternatively or additionally, the transformed and regenerated plants may also produce progeny by non-sexual propagation such as cloning, grafting. The generated transformed plants may be propagated by a variety of means, such as by clonal propagation or classical breeding techniques. For example, a first generation (or T1) transformed plant may be selfed to give homozygous second generation (or T2) transformants, and the T2 plants further propagated through classical breeding techniques.
The generated transformed organisms may take a variety of forms. For example, they may be chimeras of transformed cells and non-transformed cells; clonal transformants (e.g., all cells transformed to contain the expression cassette); grafts of transformed and untransformed tissues (e.g., in plants, a transformed rootstock grafted to an untransformed scion).
Following DNA transfer and growth of the transformed cells, putatively transformed plant cells or plants may be evaluated, for instance using Southern analysis, for the presence of the gene of interest, copy number and/or genomic organization. Alternatively or additionally, expression levels or expression patterns of the newly introduced DNA may be undertaken using northern and/or Western analysis, both techniques being well known to persons having ordinary skill in the art.
The present invention clearly extends to plants obtainable by any of the methods according to the present invention, which plants comprise any of the isolated promoters or the constructs of the present invention. The present invention clearly extends to any plant parts and propagules of such plant. The present invention extends further to encompass the progeny of a primary transformed cell, tissue, organ or whole plant that has been produced by any of the aforementioned methods, the only requirement being that progeny exhibit the same genotypic and/or phenotypic characteristic(s) as those produced in the parent by the methods according to the invention. The invention also extends to harvestable parts of a plant, such as but not limited to seeds, leaves, fruits, flowers, stem cultures, stem, rhizomes, roots, tubers, bulbs and cotton fibers.
The term "plant" or "plants" as used herein encompasses whole plants, ancestors and progeny of plants and plant parts, including seeds, shoots, stems, roots (including tubers), and plant cells, tissues and organs. The term "plant" therefore also encompasses suspension cultures, embryos, meristematic regions, callus tissue, gametophytes, sporophytes, pollen, and microspores. Plants that are particularly useful in the methods of the invention include all plants which belong to the superfamily Viridiplantae, in particular monocotyledonous and dicotyledonous plants including a fodder or forage legume, ornamental plant, food crop, tree, or shrub selected from the list comprising Acacia spp., Acer spp., Actinidia spp., Aesculus spp., Agathis australis, Albizia amara, Alsophila tricolor, Andropogon spp., Arachis spp, Areca catechu, Astelia fragrans, Astragalus cicer, Baikiaea plurjuga, Betula spp., Brassica spp., Bruguiera gymnorrhiza, Burkea africana, Butea frondosa, Cadaba farinosa, Calliandra spp, Camellia sinensis, Canna indica, Capsicum spp., Cassia spp., Centroema pubescens, Chaenomeles spp., Cinnamomum cassia, Coffea arabica, Colophospermum mopane, Coronillia varia, Cotoneaster serotina, Crataegus spp., Cucumis spp., Cupressus spp., Cyathea dealbata, Cydonia oblonga, Cryptomeria japonica, Cymbopogon spp., Cynthea dealbata, Cydonia oblonga, Dalbergia monetaria, Davallia divaricata, Desmodium spp., Dicksonia squarosa, Diheteropogon amplectens, Dioclea spp, Dolichos spp., Dorycnium rectum, Echinochloa pyramidalis, Ehrartia spp., Eleusine coracana, Eragrestis spp., Erythrina spp., Eucalyptus spp., Euclea schimperi, Eulalia villosa, Fagopyrum spp., Feijoa sellowiana, Fragaria spp., Flemingia spp, Freycinetia banksii, Geranium thunbergii, Ginkgo biloba, Glycine javanica, Gliricidia spp, Gossypium hirsutum, Grevillea spp., Guibourtia coleosperma, Hedysarum spp., Hemarthia altissima, Heteropogon contortus, Hordeum vulgare, Hyparrhenia rufa, Hypericum erectum, Hyperthelia dissoluta, Indigo incarnata, Iris spp., Leptarrhena pyrolifolia, Lespediza spp., Lettuca spp., Leucaena leucocephala, Loudetia simplex, Lotonus bainesii, Lotus spp., Macrotyloma axillare, Malus spp., Manihot esculenta, Medicago sativa, Metasequoia glyptostroboides, Musa sapientum, Nicotianum spp., Onobrychis spp., Ornithopus spp., Oryza spp., Peltophorum africanum, Pennisetum spp., Persea gratissima, Petunia spp., Phaseolus spp., Phoenix canariensis, Phormium cookianum, Photinia spp., Picea glauca, Pinus spp., Pisum sativum, Podocarpus totara, Pogonarthria fleckii, Pogonarthria squarrosa, Populus spp., Prosopis cineraria, Pseudotsuga menziesii, Pterolobium stellatum, Pyrus communis, Quercus spp., Rhaphiolepsis umbellata, Rhopalostylis sapida, Rhus natalensis, Ribes grossularia, Ribes spp., Robinia pseudoacacia, Rosa spp., Rubus spp., Salix spp., Schyzachyrium sanguineum, Sciadopitys verticillata, Sequoia sempervirens, Sequoiadendron giganteum, Sorghum bicolor, Spinacia spp., Sporobolus fimbriatus, Stiburus alopecuroides, Stylosanthos humilis, Tadehagi spp, Taxodium distichum, Themeda triandra, Trifolium spp., Triticum spp., Tsuga heterophylla, Vaccinium spp., Vicia spp. Vitis vinifera, Watsonia pyramidata, Zantedeschia aethiopica, Zea mays, amaranth, artichoke, asparagus, broccoli, brussel sprout, cabbage, canola, carrot, cauliflower, celery, collard greens, flax, kale, lentil, oilseed rape, okra, onion, potato, rice, soybean, straw, sugarbeet, sugar cane, sunflower, tomato, squash, and tea, trees and algae amongst others. According to a preferred feature of the present invention, the plant is a crop plant such as soybean, sunflower, canola, alfalfa, rapeseed, cotton, tomato, potato, tobacco, squash, papaya, poplar, leguminosa, flax, lupinus or sorghum. According to another preferred embodiment of the present invention the plant is a monocotyledonous plant, such as sugarcane, further preferable a cereal such as rice, maize, wheat, barley, millet, rye or oats.
The invention further provides a method for driving and/or regulating expression of a nucleic acid in a plant or plant cell, comprising: a) Operably linking a nucleic acid to an isolated nucleic acid according to the invention as described hereinabove, such as to any one of SEQ ID NO 1 to 22 or a variant or fragment thereof, and b) Introducing the resultant genetic construct into a plant or plant cell.
Preferably the operably linked nucleic acid of (a) is heterologous to the nucleic acids according to the present invention.
This method may further comprise cultivating the transformed plant or plant cell under conditions promoting growth, promoting regeneration and/or promoting maturation.
Furthermore, the expression of the operably linked nucleic acid may be driven and/or regulated in particular cells, tissues or organs of a plant. Accordingly, the invention provides a method as described above, wherein the expression is constitutive expression or tissue-specific expression. For these embodiments, reference is made to the example section where the specific expression patterns of the promoters according to the invention are described and where different types of tissue-specific expression are detailed.
The present invention further encompasses the use of an isolated nucleic acid as defined hereinabove to drive and/or regulate expression of an operably linked nucleic acid. (i) The person skilled in the art will recognize that provision of sequences SEQ ID NO 1 to 22, readily makes available the tools to isolate related promoters, which may have substantial sequence identity to any of SEQ ID NO 1 to 22. Additionally, provision of sequences SEQ ID NO 23 to 44 (CDS corresponding to the promoters of the present invention, see Table 1), readily makes available the tools to isolate related promoters, of which the related CDSs may have substantial sequence identity to any of SEQ ID NO 23 to 44. Therefore the present invention also encompasses a method for isolating nucleic acids, capable of driving and/or regulating expression of an operably linked nucleic acid, comprising screening a nucleic acid sequence database to find homologues of any of the sequences represented by SEQ ID NO 1 to 22 or SEQ ID NO 23 to 44. Subsequently these homologues are used to screen a library with genomic DNA, which library is for example prepared from the organism of origin of the above mentioned homologue. The screening procedure may for example involve hybridization. Subsequently, the genomic DNA that matches the homologue, is analysed to identify the transcription initiation site and the translation initiation site of the gene corresponding to the homologue. Finally, specific primers are designed for amplification of a nucleic acid located in the region upstream (at the 5' end) of said translation initiation site.
The present invention extends to the identification of regulatory proteins that are involved in the regulation of the activity of the promoters according to the present invention. Such identification may be achieved using a yeast one-hybrid system. In such a yeast one-hybrid system the sequences according to any one of SEQ ID NO 1 to 22 are operably linked to the GAL transcription activator and transformed to a yeast cell culture. That yeast cell culture is again transformed with a library of constructs encoding candidate regulatory factors.
The present invention will now be described with reference to the following figures in which:
FIG. 1 shows a general schematic representation of a promoter. Regulatory elements are sequences that may for example be responsible for special and/or temporal regulation of the promoter activity. The minimal promoter is the minimal sequence necessary and sufficient to drive expression. It includes a TATA box, which is necessary to correctly direct the RNA polymerase II to the transcription initiation site. The transcription initiation element (INR) includes the transcription initiation start site. The 5' untranslated region (5'UTR) is the region that is transcribed into pre-messenger RNA and eventually into mRNA, but is not translated into protein. The translation initiation codon is represented by the startcodon ATG.
FIG. 2 is a map of the vector p4581 useful for expression in plants of a β-glucuronidase (GUS) gene under control of any one of the promoters according to the invention. This binary vector comprises a Gateway recombination cassette, suitable for the recombination cloning of any of the promoters of the present invention in front of the Escherichia coliβ-glucuronidase (GUS) gene. This cassette contains a chloramphenicol resistance gene (CamR) and the ccdB suicide gene for counter selection of non-recombined plasmids, This GUS expression cassette further comprises the double terminator sequence T-zein and T-rbcS-deltaGA. This expression cassette is located within the left border (LB repeat, LB Ti C58) and the right border (RB repeat, RB Ti C58) of the nopaline Ti plasmid. Cloned within these borders are also selectable marker and a screenable marker genes each under control of a constitutive promoter and a terminator sequence. This vector also contains an origin of replication (pBR322) for bacterial replication and a bacterial selectable marker (Spe/SmeR) for bacterial selection.
The following figures show the results of the GUS staining of plants or plant parts transformed with the reporter vector p4581 carrying a promoter according to the present invention operably linked to the reporter gene GUS. Plants denoted "C plants" are transgenic plants grown to about 5 cm; Plants denoted "B plants" are grown to about 10 cm; and plants denoted "A plants" are grown to maturity. These A plants were used to collect different tissue samples from old leaves, young leaves and seeds.
FIG. 3 shows the expression pattern of PRO0110 (RCc3, SEQ ID NO 1). GUS staining is visible in roots.
FIG. 4 shows the expression pattern of PRO0005 (putative beta-amylase, SEQ ID NO 2). GUS staining is visible in seeds, more specifically in the embryo or in the scutellum of the embryo.
FIG. 5 shows the expression pattern of PRO0009 (putative cellulose synthetase, SEQ ID NO 3). GUS staining is visible in roots.
FIG. 6 shows the expression pattern of PRO0058 (proteinase inhibitor Rgpi9, SEQ ID NO 4). GUS staining is visible in the seeds.
FIG. 7 shows the expression pattern of PRO0061 (beta expansine EXPB9, SEQ ID NO 5). GUS staining is visible in young flowers of A plants (A) and in other young expanding tissues of B plants (B) and C plants (C).
FIG. 8 shows the expression pattern of PRO0063 (putative structural protein, SEQ ID NO 6). GUS staining is visible in young tissues, for example in the calli (A) or old leaves, young leaves and seeds of "A plants" (B).
FIG. 9 shows the expression pattern of PRO0081 (putative caffeoyl-CoA 3-O-methyltransferase, SEQ ID NO 7). GUS staining is visible in young tissues, particularly of the shoot.
FIG. 10 shows the expression pattern of PRO0091 (prolamine RP5, SEQ ID NO 8). GUS staining is visible in seeds (A), particularly in the endosperm, and in meristem (B).
FIG. 11 shows the expression pattern of PRO0095 (putative amino peptidase, SEQ ID NO 9). GUS staining is visible in seeds, more particularly in the embryo.
FIG. 12 shows the expression pattern of PRO0111 (uclacyanin 3-like protein, SEQ ID NO 10). GUS staining is visible in roots and in meristem.
FIG. 13 shows the expression pattern of PRO0116 (26S proteasome regulatory particle non-ATPase subunit 11, SEQ ID NO 11). GUS staining is weakly visible in the whole plant (weak constitutive) and is particularly visible in meristem.
FIG. 14 shows the expression pattern of PRO0117 (putative 40S ribosomal protein, SEQ ID NO 12). GUS staining is visible in the seeds, more particularly in the endosperm.
FIG. 15 shows the expression pattern of PRO0122 (chlorophyll a/b-binding protein presursor (Cab27), SEQ ID NO 13). GUS staining is visible in the shoot.
FIG. 16 shows the expression pattern of PRO0123 (putative protochlorophyllide reductase, SEQ ID NO 14). GUS staining is visible in the shoot (above-ground tissues).
FIG. 17 shows the expression pattern of PRO0133 (chitinase Cht-3, SEQ ID NO 15). GUS staining is visible in the roots and meristem.
FIG. 18 shows the expression pattern of PRO0151 (WSI18, SEQ ID NO 16). GUS staining is visible in the calli and upper plant parts (A) as well as in the aleurone layer and embryo (B).
FIG. 19 shows the expression pattern of PRO0169 (aquaporine, SEQ ID NO 17). GUS staining is visible in the whole plant (constitutive expression).
FIG. 20 shows the expression pattern of PRO0170 (High mobility group protein, SEQ ID NO 18). GUS staining is strongly visible in the whole plant as is illustrated by the "B plants" (A), and various tissues such as old leaves, young leaves and seeds (B) and calli (C) (constitutive expression).
FIG. 21 shows the expression pattern of PRO0171 (reversibly glycosylated protein RGP1, SEQ ID NO 19). GUS staining is visible in all plant parts (constitutive expression).
FIG. 22 shows the expression pattern of PRO0173 (cytosolic MDH, SEQ ID NO 20). GUS staining is visible in all plant parts and particularly in the shoot (above-ground tissues) and seeds.
FIG. 23 shows the expression pattern of PRO0175 (RAB21, SEQ ID NO 21). GUS staining is weakly visible in calli (A), meristems and young leaves, and is strongly visible in developing and maturing seeds (B) more particularly in the embryo.
FIG. 24 shows the expression pattern of PRO0177 (Cdc2-1, SEQ ID NO 22). GUS staining is weakly visible in meristem and in leaf sheets.
The promoters according to the present invention were isolated as DNA regions spanning about 1.2 kb of the sequence upstream of the translation initiation codon (i.e. first ATG, which codon was excluded) from various rice genes. For determination of their nucleic acid sequence and their expression pattern, the following procedure was followed: First in silico studies on genomic rice sequences were performed. However, procedures based on automated prediction programs to locate promoter-like nucleic acid sequence are highly error prone, even for the localization the best-characterized promoter control elements such as the TATA box and the transcription initiation element (INR). Also, in silico determination of expression pattern is extremely speculative. Therefore, to obtain unambiguous data about the nucleic acid sequence and the expression pattern of the promoters, in vivo studies were performed encompassing (i) isolation of the promoter nucleic acid sequence; (ii) operably linking a reporter gene to the promoter and introducing the resulting genetic construct into a host organisms; (iii) growing the transformed host cell under conditions allowing expression of the reporter gene, and (iv) determination of the reporter gene activity in the different tissues of the host organism. These methods are now described in more detail.
Identification and Isolation of the Promoters
Identification of Rice ESTs, the Corresponding Genes and their Location in the Rice Genome
Sequence databases, comprising rice sequences, were searched for rice expressed sequence tags (ESTs). Subsequently an "in silico" Northern-blot was performed to allow identification of EST families that are strongly expressed or that are specific for a particular organ. This analysis included normalization of the numbers of ESTs isolated from different plant organs. The ESTs families with an interesting distribution among source cDNA libraries were selected for further analysis and sequence homology searches. After sequence homology searches in combination with scanning scientific data, the genes that correspond to those families of ESTs were identified from sequence databases and a (putative) function and corresponding gene name was given (see Table 1). Subsequently, the corresponding promoter region was isolated by the following procedure. In a first step the TIGR database was searched to find a tentative contig corresponding to an EST family. Sequence homology was found using standard computer programs, such as Blast N using standard parameters (typically G Cost to open a gap=5, E Cost to extend a gap=2, q Penalty for a mismatch in the blast portion of run=-3, r Reward for a match in the blast portion of run=1, e Expectation value=10.0, W Word size=11, v Number of one-line descriptions=100, b Number of alignments to show=100, Matrix=BLOSUM62). The TIGR database (The Institute for Genomic Research), provides Tentative Contigs (TC) which are sequence predictions based on contig building from all known EST, from all known cDNA and from reconstructed mRNA. The TCs used for identification of the promoters of the present invention are represented in Table 1. In a second step these TCs were used to locate the corresponding gene on a genomic sequence, which gene comprises the coding region as well as the promoter region. Generally, these genomic sequences were BAC clones, which are represented herein by their Genbank accession number (see Table 1). From these BAC clones the sequence identity of the promoter region could be determined.
TABLE-US-00001 TABLE 1 list of rice promoters of the present invention. The promoter sequences are represented herein by their SEQ ID NO and promoter number (PRO). The coding sequences (CDS) naturally driven by a promoter of the present invention are represented by their name, by SEQ ID NO and by Tentative contig (TC) accession number of the TIGR database. The Genomic sequences (BAC clones or genes) comprising a promoter region of the present invention are represented by their Genbank accession number. Prom Prom CDS BAC clone SEQ ID NO number CDS name SEQ ID NO CDS TC (*or gene) 1 PRO0110 RCc3 23 TC89946 AC037426 2 PRO0005 putative beta-amylase 24 TC90358 AC022457 3 PRO0009 putative cellulose synthase 25 TC83635 AC022457 4 PRO0058 proteinase inhibitor Rgpi9 26 TC83117 AF044059 5 PRO0061 beta expansine EXPB9 27 TC89913 AC020666 6 PRO0063 structural protein 28 TC89985 AP001278 7 PRO0081 putative caffeoyl-CoA 3-O- 29 TC89891 AP000364 methyltransferase 8 PRO0091 prolamine RP5 30 TC89670 AF156714* 9 PRO0095 putative methionine aminopeptidase 31 TC89883 AC027133 10 PRO0111 uclacyanin 3-like protein 32 TC90434 AJ307662 11 PRO0116 26S proteasome regulatory particle 33 TC83072 AP000969 non-ATPase subunit 11 12 PRO0117 putative 40S ribosomal protein 34 TC90038 AC090871 13 PRO0122 chlorophyll a/b-binding protein presursor 35 TC82936 AP004700 (Cab27) 14 PRO0123 putative protochlorophyllide reductase 36 TC89839 AL606456 15 PRO0133 chitinase Cht-3 37 TC85888 D16223* 16 PRO0151 WSI18 38 TC84300 AP003023 17 PRO0169 aquaporine 39 TC89687 AP005108 18 PRO0170 High mobility group protein 40 TC89846 AP004004 19 PRO0171 reversibly glycosylated protein RGP1 41 TC82935 AC090874 20 PRO0173 cytosolic MDH 42 TC82977 AC037425 21 PRO0175 RAB21 43 TC83646 Y00842* 22 PRO0177 Cdc2-1 44 TC90619 AP004765
Identification and Isolation of the Promoter Regions of Rice Genes
Starting from the sequence information of the genes and their location in the rice genome, the promoter regions of these genes were isolated as the DNA region spanning about 1.2 kb upstream of the translation initiation codon (i.e. first ATG), which codon was excluded. When an intervening sequence such as an intron, was present in the 5' untranslated region of the gene, the isolated DNA region was taken as the region spanning about 1.2 kb plus the length of that intervening sequence. The promoter regions were isolated from genomic DNA of Oryza sativa Japonica or exceptionally from Oryza sativa Indica via PCR using specific primers. These specific primers comprise AttB recombination sites, suitable for recombination cloning of the isolated promoter region These specific primers are herein represented as SEQ ID NO 45 to 88 and are listed in Table 2. Conditions for PCR were as follows: 1 cycle of 2 min at 94° C., 35 cycles of 1 min at 94° C., 1 min at 58° C. and 2 min at 68° C., and 1 cycle of 5 min at 68° C. The length of the expected PCR fragment is also indicated in Table 2. The corresponding PCR fragment was purified from the PCR reaction mix via gele electrophoresis and subsequent purification using Zymoclean Gel DNA Recovery Kit (Zymo Research, Orange, Calif.).
TABLE-US-00002 TABLE 2 Overview of the primers used to isolate the rice promoters of the present invention and the length of the rice promoter regions. Promoter Promoter Prom Primer forward Primer Primer reverse Primer SEQ ID NO number length SEQ ID NO forward SEQ ID NO reverse 1 PRO0110 1264 45 prm3780 67 prm3781 2 PRO0005 1215 46 prm2768 68 prm2769 3 PRO0009 1038 47 prm2420 69 prm2421 4 PRO0058 1301 48 prm2853 70 prm2854 5 PRO0061 1243 49 prm2426 71 prm2427 6 PRO0063 1019 50 prm2855 72 prm2856 7 PRO0081 1212 51 prm3025 73 prm3026 8 PRO0091 1052 52 prm3029 74 prm3030 9 PRO0095 1216 53 prm3061 75 prm3062 10 PRO0111 1237 54 prm3031 76 prm3032 11 PRO0116 1100 55 prm3051 77 prm3052 12 PRO0117 1216 56 prm3592 78 prm3049 13 PRO0122 1210 57 prm5131 79 prm2195 14 PRO0123 123 58 prm3782 80 prm2197 15 PRO0133 1808 59 prm2844 81 prm2845 16 PRO0151 1828 60 prm2973 82 prm2974 17 PRO0169 1267 61 prm3770 83 prm3771 18 PRO0170 1130 62 prm3772 84 prm3773 19 PRO0171 1230 63 prm3774 85 prm3775 20 PRO0173 1234 64 prm3776 86 prm3777 21 PRO0175 1553 65 prm3800 87 prm3801 22 PRO0177 1087 66 prm5135 88 prm5136
Cloning of Promoter-GUS Reporter Vectors for Plant Transformation
The purified PCR fragments of Example 1, corresponding to the promoter regions of the present invention, were cloned into the pDONR201 entry plasmid of the Gateway® system (Life Technologies) using the "BP recombination reaction". The identity and base pair composition of the cloned insert was confirmed by sequencing and additionally, the resulting plasmid was tested via restriction digests.
In order to clone each of the promoters of the present invention in front of a reporter gene, each entry clone of Example 1 was subsequently used in an "LR recombination reaction" (Gateway®) with the destination vector p4581. This destination vector was designed to operably link each promoter of the present invention to the Escherichia coli beta-glucuronidase (GUS) gene via the substitution of the Gateway recombination cassette in front of the GUS gene. Furthermore this destination vector is suitable for transformation of plants and comprises within the T-DNA left and right borders the resulting promoter-GUS cassette and selectable marker and screenable marker cassettes (see FIG. 2). The resulting reporter vectors, comprising a promoter of the present invention operably linked to GUS, are subsequently transformed into Agrobacterium strain LBA4044 and subsequently into rice plants using standard transformation techniques.
Expression Patterns of the Promoter-GUS Reporter Cassette in Plants
Growth and Harvest of Transgenic Plants or Plant Parts at Various Stages (C Plants, B Plants and a Plants)
For each promoter-GUS reporter construct, 3 T0 transgenic rice plants were generated from transformed cells. Plant growth was performed under normal conditions. The first transgenic plant was sacrificed for GUS staining when it had reached a size of about 5 cm, which plant is named herein "C plant". The second transgenic plant was sacrificed for GUS staining when it had reached a size of about 10 cm, which plant is named herein "B plant". The third transgenic plant was kept for seed production and is named herein "A plant". GUS staining was performed on complete C and B plants. On A plants, GUS staining was performed on leaf pieces, flowers and section of seeds at various developmental stages. A plants were allowed to set seed, which seeds were used after harvest for confirmation of the expression pattern in T1 plants.
The sacrificed plants or plant parts were covered with 90% ice-cold acetone and incubated for 30 min at 4° C. After 3 washes of 5 min with Tris buffer [15.76 g Trizma HCl (Sigma T3253)+2.922 g NaCl in 1 l bidi, adjusted to pH 7.0 with NaOH], the material was covered by a Tris/ferricyanate/X-Gluc solution [9.8 ml Tris buffer+0.2 ml ferricyanate stock (0.33 g Potassium ferricyanate (Sigma P3667) in 10 ml Tris buffer)+0.2 ml X-Gluc stock (26.1 mg X-Gluc (Europa Bioproducts ML 113A) in 500 μl DMSO)]. Vacuum infiltration was applied for 15 to 30 minutes. The plants or plant parts were incubated for up to 16 hours at 37° C. until development of blue colour was visible. The samples were washed 3 times for 5 minutes with Tris buffer. Chlorophyll was extracted in ethanol series of 50%, 70% and 90% (each for 30 minutes).
Expression Patterns of the Promoters of the Present Invention
The expression patterns of the rice promoters of the present invention are summarized in Table 3.
TABLE-US-00003 TABLE 3 expression patterns of the rice promoters of the present invention PRO Promoter SEQ ID NO number Promoter name Expression pattern 1 PRO0110 RCc3 strong root 2 PRO0005 putative beta-amylase Embryo (scutellum) 3 PRO0009 putative cellulose synthase weak in roots 4 PRO0058 proteinase inhibitor Rgpi9 seed 5 PRO0061 beta expansine EXPB9 weak in young tissues 6 PRO0063 structural protein young tissues + calli + embryo 7 PRO0081 putative caffeoyl-CoA 3-O- shoot methyltransferase 8 PRO0091 prolamine RP5 meristem + strong in endosperm 9 PRO0095 putative methionine aminopeptidase embryo 10 PRO0111 uclacyanin 3-like protein weak meristem 11 PRO0116 26S proteasome reg. particle weak meristem non-ATPase s.u. 11 12 PRO0117 putative 40S ribosomal protein weak in endosperm 13 PRO0122 chlorophyll a/b-binding protein weak in shoot presursor (Cab27) 14 PRO0123 putative protochlorophyllide reductase strong shoot specific 15 PRO0133 chitinase Cht-3 weak meristem specific 16 PRO0151 WSI18 Calli + shoot + strong embryo 17 PRO0169 aquaporine medium constitutive 18 PRO0170 High mobility group protein strong constitutive 19 PRO0171 reversibly glycosylated protein RGP1 weak constitutive 20 PRO0173 cytosolic MDH Shoot and seed 21 PRO0175 RAB21 embryo 22 PRO0177 Cdc2-1 weak in meristem + strong seed
The following paragraphs describe the observed expression patterns of the promoters of the present invention in more detail. The observations are based on the visual inspection of the GUS stained tissues as described above. It is to be understood that for some promoters expression may be weak and that expression in certain tissues may only be visible with very sensitive detection methods.
PRO0110--SEQ ID NO 1--RCc3
1 construct (OS1432), which is a reporter vector as described in Example 2 comprising PRO0110 was investigated. 25 calli, 14 C, 21 B plants and 21 A plants were analysed. There was no expression visible in calli, but strong expression in roots of C plants (93%) and of B plants (81%) was observed. No expression in the shoots of A plants was observed. Therefore the RCc3 promoter PRO0110 is suitable for strong expression in roots.
PRO0005--SEQ ID NO 2--Putative Beta-Amylase
1 construct (OS1365) was investigated. 28 calli, 24 B plants and 22 A plants were analysed. Occasional expression in calli (7%) was observed as well as occasional weak expression in roots (4%) and shoots (12%) of B plants, expression in the scutellum of embryos of A plants (43%) and occasional expression in leaves (5%) of A plants. This promoter is therefore suitable for expression in embryo, more preferably in the scutellum of the embryo. This region of the embryo is also referred to as the transfer layer of the embryo. This promoter may have some leakiness in other tissues.
PRO0009--SEQ ID NO 3--Putative Cellulose Synthase
1 construct (OS1461) was investigated. 20 calli, 20 C, 20 B plants and 20 A plants were analysed. Occasional expression in calli (20%) was observed as well as weak expression in roots (55%) of C plants, occasional expression in young leaves (10%) of C plants and weak expression in the roots (25%) of B plants. No expression in leaves of A or B plants was observed. Therefore this promoter is suitable for expression in roots. This promoter may show some leakiness in the leaves.
PRO0058--SEQ ID NO 4--Proteinase Inhibitor Rgpi9
1 construct (OS1370) was investigated. 13 B plants and 12 A plants were analysed. No expression was observed in B plants. In A plants, no expression was observed in the leaves, but there was strong expression in endosperm and embryo (58-42%). Therefore, this promoter PRO0058 is suitable for expression in seeds.
PRO0061--SEQ ID NO 5--Beta Expansine EXPB9
2 constructs (OS1441 and OS1460) were investigated. 20 calli, 32 C, 32 B plants and 32 A plants were analysed. Weak expression was observed in the leaves of C and B plants. In A plants expression in the flowers was observed (44%), more particularly in lemma of young spikelets. It was concluded that the promoter PRO0061 is suitable for expression in young tissue, more preferably in young, developing or expanding tissue, more preferably in green tissue.
PRO0063--SEQ ID NO 6--Putative Structural Protein
1 construct (OS1446) was investigated. 13 calli, 13 C, 13 B plants and 12 A plants were analysed. In calli, weak expression was detected (92%). In C plants, there was no expression in roots and there was weak expression in some leaves (46%). In B plants, there was no expression in roots and weak expression in young tillers (78%) or young leaves (54%), but no expression in old leaves. In A plants, there was occasional expression in young leaves (17%) and expression in embryo and scutellum (42%). Therefore it was concluded that this promoter is active in the above-ground tissues, such as leaf, stem and seed. These data demonstrate that the promoter is suitable for expression in calli and in the shoot, and for expression in young tissues and seeds.
PRO0081--SEQ ID NO 7--Putative Caffeoyl-CoA 3-O-Methyltransferase
1 construct (OS1419) was investigated. 20 calli, 20 C, 20 B plants and 20 A plants were analysed. No expression was observed in Calli. Expression was observed in C plants, more particularly weak expression in root cylinder (40%) and weak expression in young leaves (80%) and in old leaves. Expression was also observed in B plants, more particularly weak expression in roots (25%) and weak expression in young leaves (80%). Expression was also observed in young leaves (50%) of A plants. It was concluded that promoter PRO0081 is suitable for expression in above-ground tissues, preferably in the shoot. This promoter may have some leakage of expression in roots.
PRO0091--SEQ ID NO 8--Prolamine RP5
1 construct (OS1558) was investigated. 12 C, 12 B plants and 12 A plants were analysed. Weak expression was observed in the discrimination centre (50%) of C plants and in the discrimination centre (58%) of B plants. Strong expression was observed in endosperm (55%) of A plants. This promoter was found to be useful for strong expression in the endosperm, with leakiness in meristem, preferably the shoot meristem or discrimination centre.
PRO0095--SEQ ID NO 9--Putative Methionine Aminopeptidase
1 construct (OS1423) was investigated. 16 calli, 14 C, 14 B plants and 16 A plants were analysed. Some expression was observed in root-tips (36%) of C plants and in the embryo (38%) of A plants, but not in endosperm of A plants. It was concluded that PRO0095 is suitable for expression in embryo.
PRO0111--SEQ ID NO 10--Uclacyanin 3-Like Protein
1 construct (OS1421) was investigated. 22 calli, 21 C, 22 B plants and 21 A plants were analysed. Weak expression was observed in the discrimination centre and meristems (77%) of B plants. It was concluded that promoter PRO0111 is suitable for weak expression in the meristem, preferably in shoot meristem or discrimination centre.
PRO0116--SEQ ID NO 11--26S Proteasome Regulatory Particle Non-ATPase Subunit 11
1 construct (OS1679) was investigated. 13 C, 14 B plants and A plants were analysed. Weak expression was observed in meristem/discrimination centre of C plants (38%) and of B plants (71%) and in young leaf sheaths of C plants (77%) and of B plants (21%). It was concluded that promoter PRO0116 is suitable for expression in meristem, preferably in shoot meristem or discrimination centre.
PRO0117--SEQ ID NO 12--Putative 40S Ribosomal Protein
1 construct (OS1425) was investigated. 9 calli, 9 C, 9 B plants and 9 A plants were analysed. Occasional weak expression was observed in roots (22%) and in young leaf blades (44%) of C plants. Expression was mainly observed in endosperm (37%) of A plants. Therefore, promoter PRO117 was found to be suitable for expression in endosperm and may have some leakiness in young leaves.
PRO0122--SEQ ID NO 13--Chlorophyll a/b-Binding Protein Presursor (Cab27)
1 construct (OS1675) was investigated. 38 calli, 38 C, 38 B plants and 15 A plants were analysed. Very weak expression was observed in the discrimination centre and young leaf sheaths of C plants. It was concluded that this promoter PRO0122 is suitable for weak expression in shoots.
PRO0123--SEQ ID NO 14--Putative Protochlorophyllide Reductase
1 construct (OS1433) was investigated. 21 calli, 18 C, 19 B plants and 18 A plants were analysed. Strong expression was observed in shoots (33-68%) of C plants and B plants (63-79%). In B plants there was also occasional expression in roots. In A plants, again strong expression in young leaves (73%) was observed, as well as occasional expression in old leaves (39%). It was concluded that this promoter is suitable for strong expression in shoots, preferably in leaves.
PRO0133--SEQ ID NO 15--Chitinase Cht-3
1 construct (OS1687) was investigated. 15 calli, 12 C, 16 B plants and 12 A plants were analysed. Weak expression was observed in calli (66%) and in the discrimination centre/meristem (50%) of B plants. It was concluded that promoter PRO0133 is suitable for weak expression in meristem, preferably in shoot meristem or discrimination centre.
PRO0151--SEQ ID NO 16--WSI18
1 construct (OS1458) was investigated. 22 calli, 16 C, 16 B plants and 13 A plants were analysed. Strong expression was observed in calli (91%) and weak expression in shoots of C plants (62%). In A plants there was very strong expression in the aleurone layer and in the embryo (46%). It was concluded that promoter PRO0151 is suitable for strong expression in calli and in seeds, more particularly in the aleurone layer and in the embryo of the seeds.
PRO0169--SEQ ID NO 17--Aquaporine
1 construct (OS1911) was investigated. 11 calli, 10 C plants, B plants and A plants were analysed. Some expression (55%) was observed in calli and in roots (30%) of C plants. Furthermore, good expression was observed in shoot tissues (80%) of C plants and in young leaves of B plants. It was concluded that this promoter is suitable for constitutive expression, preferably constitutive in young plants.
PRO170--SEQ ID NO 18--High Mobility Group Protein
1 construct (OS1434) was investigated. 23 calli, 21 C, 21 B plants and 14 A plants were analysed. Expression was observed in calli (52%) and in roots (51%) of C plants. Moreover, strong expression was observed in young leaves (81%) of C plants, in roots (86%) of B plants and in young leaves (86%) of B plants. In A plants there was strong expression in young leaves (75%), old leaves (43%), embryo and aleurone but a weaker expression in endosperm (82%). It was concluded that promoter PRO170 is suitable for strong constitutive expression.
PRO0171--SEQ ID NO 19--Reversibly Glycosylated Protein RGP1
1 construct (OS1762) was investigated. 18 calli, 11 C and 13 B plants were analysed. Strong expression was observed in calli (44%) and in all tissues (27%) of C plants. In all tissues of B plants (16%), expression was somewhat weaker but most pronounced the in discrimination centres (46%). It was concluded that promoter PRO0171 is suitable for constitutive expression.
PRO0173--SEQ ID NO 20--Cytosolic MDH
1 construct (OS1435) was investigated. 17 calli, 17 C, 17 B plants and 15 A plants were analysed. Occasional expression (12%) was observed in calli and weak expression was observed in upper parts (24-69%) of C plants as well as in young leaves (41%) of B plants. In A plants, expression in leaves (33%) was observed and strong expression in seeds (38%), but not in the root. It was concluded that the promoter PRO0173 is suitable for expression in above-ground tissues especially for constitutive expression in the shoot and especially in the seeds.
PRO0175--SEQ ID NO 21--RAB21
1 construct (OS1436) was investigated. 16 calli, 12 C, 15 B plants and 15 A plants were analysed. Expression was observed in some calli (31%), in the discrimination centres (42%) of C plants and in young leaves (25-58%) of C plants and A plants (15%). Furthermore, very strong expression was observed in aleurone and embryo (60%) of a plant. It was concluded that promoter PRO0175 is suitable for strong expression in calli and in seeds, more particularly in developing/maturing seeds, more particularly in the aleurone layer and in the embryo of the seeds.
PRO0177--SEQ ID NO 22--Cdc2-1
1 construct (OS1436) was investigated. 16 calli, 12 C, 15 B plants and 15 A plants were analysed. Expression was observed in some of the calli (31%), in the discrimination centre (42%) of C plants, in young leaves (25-58%) of C plants and occasionally in young leaves (15%) of A plants. Moreover, very strong expression was observed in aleurone and embryo (60%) of seeds from A plants. It was concluded that this promoter is suitable for specific expression in seeds, more particularly in developing/maturing seeds.
Stability of the Expression Patterns of the Promoters of the Present Invention in Further Generations
The above-mentioned analyses were performed on T0 plants originating from the transformed tissues. The stability of promoter activity in the next generations or progeny plants of the original T0 plant, the so-called T1 and T2 plants, was evaluated as follows. The T0 plant transformed with the reporter constructs as mentioned in the above paragraphs of Example 2, were grown until maturity (A plants), of which the seeds (T1 seeds) were harvested and sown to generate progeny T1 plants. These plants were analysed as described above in Example 3 and the A T1 plants were allowed to reach maturity and to set T2 seeds.
The expression pattern of the promoters of the present invention was studied in T0 plants, T1 seeds, T1 plants and T2 seeds and in all the tissues (including seeds and seed tissues) as described in Example 3. The specific expression patterns as reported from the T0 and T1 seeds and described in Example 3 were confirmed in the following T1 generation and T2 seeds. It is concluded that the expression pattern of the promoters of the present are stably inherited in plants of subsequent generations.
Stability of Expression Patterns of the Promoters of the Present Invention in Other Plants
The above-mentioned plant analyses were performed on rice plants. This choice was based on the practical consideration that plant genetic engineering is most profitable for crop plants. Also in other crop plants, such as for example Zea Mays, the reporter constructs comprising the promoters according to the present invention are introduced and transformed plant are evaluated as described hereinabove. The expression patterns of the promoters according to the present invention are conserved among plants. Therefore, the promoters according to the present invention are also suitable for driving and/or regulating expression of an operably linked nucleic acid in monocots, such as corn.
For many other purposes such as research and horticulture, (small) herbs are being genetically modified, which involves the use of promoters. Therefore the reporter constructs comprising the promoters according to the present invention are introduced into other plants species such as for example Arabidopsis thaliana and transformed plants are evaluated as described hereinabove. The expression patterns of the promoters according to the present invention are conserved among plants. Therefore, the promoters according to the present invention are also suitable for driving and/or regulating expression of an operably linked nucleic acid in other plant species such as for example dicots, such as Arabidopsis.
8811264DNAOryza sativamisc_featurePRO0110 - RCc3 1tcgacgctac tcaagtggtg ggaggccacc gcatgttcca acgaagcgcc aaagaaagcc 60ttgcagactc taatgctatt agtcgcctag gatatttgga atgaaaggaa ccgcagagtt 120tttcagcacc aagagcttcc ggtggctagt ctgatagcca aaattaagga ggatgccaaa 180acatgggtct tggcgggcgc gaaacacctt gataggtggc ttacctttta acatgttcgg 240gccaaaggcc ttgagacggt aaagttttct atttgcgctt gcgcatgtac aattttattc 300ctctattcaa tgaaattggt ggctcactgg ttcattaaaa aaaaaagaat ctagcctgtt 360cgggaagaag aggattttgt tcgtgagaga gagagagaga gagagagaga gagagagaga 420gaaggaggag gaggattttc aggcttcgca ttgcccaacc tctgcttctg ttggcccaag 480aagaatccca ggcgcccatg ggctggcagt ttaccacgga cctacctagc ctaccttagc 540tatctaagcg ggccgaccta gtagccacgt gcctagtgta gattaaagtt gccgggccag 600caggaagcca cgctgcaatg gcatcttccc ctgtccttcg cgtacgtgaa aacaaaccca 660ggtaagctta gaatcttctt gcccgttgga ctgggacacc caccaatccc accatgcccc 720gatattcctc cggtctcggt tcatgtgatg tcctctcttg tgtgatcacg gagcaagcat 780tcttaaacgg caaaagaaaa tcaccaactt gctcacgcag tcacgctgca ccgcgcgaag 840cgacgcccga taggccaaga tcgcgagata aaataacaac caatgatcat aaggaaacaa 900gcccgcgatg tgtcgtgtgc agcaatcttg gtcatttgcg ggatcgagtg cttcacagct 960aaccaaatat tcggccgatg atttaacaca ttatcagcgt agatgtacgt acgatttgtt 1020aattaatcta cgagccttgc tagggcaggt gttctgccag ccaatccaga tcgccctcgt 1080atgcacgctc acatgatggc agggcagggt tcacatgagc tctaacggtc gattaattaa 1140tcccggggct cgactataaa tacctcccta atcccatgat caaaaccatc tcaagcagcc 1200taatcatctc cagctgatca agagctctta attagctagc tagtgattag ctgcgcttgt 1260gatc 126421215DNAOryza sativamisc_featurePRO0005 - putative beta-amylase 2cccgatttag tagaccacat tttggcatca aaccaaaata gaccctctcc cagaatttgt 60aaatggcttt gtggttcgtg atatcactga acctgctggg tgaataaagt aaaaaaaaaa 120acccataaat tggccttctg caagatctcg tcgtcttgcc caaactatag ccttcgatct 180ttccatcagg accgcatggg gggagagcag gggcaagtat gaaatggagt tcagattcag 240attctagaac agtctgaaca tgcgacgacg acgatggcga tgtatctgaa caatctggtc 300ctctccctct cctcccgggc gggcttccac gcggctgagt ttcaggctcc caatctgcag 360ctcctcccag aaccttactc tgattgattg gttcatcgtt tccatggctc caatgaatgc 420aacgtgttgt tcagattttc tgaatcttgt tctcaatccg gagtacgtgc tgtagcagca 480gcaatctgtc cctgatctga gaattttaga cactcgtaga ttcgctgatc aatcattccg 540tcccttcgag tggtctagat tgagcttaat catcctgcta ctcgaatcaa atcttcagca 600agtgagagct agataattca gaagaaatca acatattctt cgcgaaaaaa agaaataacc 660gatgaaacca cggtaattag gttcttcgaa tcaccgggag agtaggaaaa aacgagctaa 720aatcccacat aggaggaaac ggttaaaaac ggccactccg cgtctccgcc gcgagactag 780ctctcgccag tccacgtagc ccaatccaca accgccacgt gctccgacaa tcccgcccgt 840ccatcgccgc ggccccggcc tcatctcgac cactcgtttc ctcccttcac accagccacg 900tggcactctc tcgagagctc ccgcccgcct atataaactt gttcgcgctc ggctcctcct 960cctcatcgac ctccacccca cattgaataa ttatttttaa taattttagt tttttttttg 1020gctttagata tattcccaat ccccaacctc ccaataatcc gatctctccc agttctgttc 1080ggatcaaggc tgtgtcgatc gcaaaaaaga aaaaaaaaac aatttccttt tggggtggtt 1140catctgttga tcacttcttt gtttcccgcg ttttgttggg gattcgattt tcgggttaag 1200attttctaca cgacc 121531038DNAOryza sativamisc_featurePRO0009 - putative cellulose synthase 3gccatcgagt ggtgtgccga taccggcgcc tgttctttac agcctcagct agtgttgttg 60tccgaggcaa tttttccgac ctattgtgtt gctttcctct ctgatagctt atggtaaaag 120atacaaagat gttgaggagt ttgtacgcca cttaattttg ctcgtaacat acattgacaa 180tcaagaggag ccatggcatt gcgatctgct tacacggcat attcttactg gatggtgtac 240actacttacc ctttttaatg caagcatcaa tccattgctt ttctcactgc acacctgatt 300cgtactgaaa acgtgaaaca taaaaaaaaa acaaaaatct agctgatgtt ggctctcggg 360gcctcgagtc tagtttgtcc tagatggcta acctgatatg tgttggtcac gctcacgttt 420gaaccgagaa agagtgtgtg tgtgtgtgtg tcggcgtgct gctacaccag agcctccctg 480aatcgcaatg cgtgttaacg ccagcatcgc aggatttcat ctcacttgac aggttcagat 540ggccttcctc ctaccgtctg ccatttatac acgcagtgac ttaacgctta cacgagccgg 600atggcccgga tctcccccct gcaccatctc accagaaaaa cggtgaggcg tcaccgcaac 660ccacccacca aacacatcca cgtcccttca ccgttggcct tcgattttgc ttcagctgca 720ctacgacccc tccaacacat ttccctcgcg tctcgttgcg atctcacctt acgacgatct 780cgttccagca gcagcagcat cggcagcggc ggcttgcttc cgaagcgagc aatgcatggc 840gcgcgcggcc gcgtgcgtgc gtgccttggc ttgcgctcta atcaaaccgg gacgccccaa 900ctcacggttg gtgcgggacg ccaccccgcc accttaccgc ccccgcctcc ctgcatctga 960tcatcaacca gctgctatat cacctagcta gccgccgcct cctcctcgcc caccaacgtc 1020gcttccccgg cacctcac 103841301DNAOryza sativamisc_featurePRO0058 - proteinase inhibitor Rgpi9 4tctcttctga agctgaagcc ctgcgaaata ggcctttaaa cgctttaagg ttactggatg 60atcatatcgg cgtaagaccg gtttaaacat ggtttcgctt tgtgaatcca atgtgagtca 120cgacgtgaca catggcacgt ccttggagct ttagacatat cgaatctgag cactggagtg 180gccgagtggg tgagcggcca aatccgtttt agacagatcg cactgacacg atgttgatca 240ttgatactaa taccatttta tcaagcagta gtgttgaaaa aaaaacttat gttctcttca 300actgtgagat ttcatcccgt ttcaagatga acaagccatg catgtgagat gtgaacagaa 360ggcagaagac agtggaaaga caggacaaat aagtgaagag ggatcaaatc aatgggcctg 420acggtttctg aaagttgaca tggaaatcgc cggtgatcac cggtttatac gttatttaaa 480tctgcgattt ccactttcgt ttgctttcgg ggttccaatt tgagtcacgc acatattctt 540catcgtgctt tggatctcag caccgtagta acttttggac aaattgcatt cgccgacact 600aataacatgt tctttttatg ctgctttaca tatactgctt atccacaccc aatcccatgt 660tcatatatta tgagatggag ggagtaaact ttgttaacag caacattttt tatattaaag 720catcaactaa ttaaagcaca agatacgcat gttatctcaa taaatcttcc agtgcatgta 780taaagaagat gtcgccgcta acttagataa tttttgtgac ttttatcctg gccggcataa 840ttaattcttc cggaaattaa aagctagttt ttccatattc atcagtacag acaagacagc 900atagtaagcg aagcatacct gacgtgttag ctcattgtaa ctcgatctgg aacactcgat 960gctagataca gacagacact cctcgtgatg aacgttagca tttagcaaca tacggtgata 1020aagcagctgg ggatcgatcc atccatccat cgtctttaca cgtacttacc ttgctaaccg 1080cactgtcgac tcttgcatgt ttgcatgtaa tccaaatgga ccccacgtgg aacatgctca 1140cagtgctttg cagctgcttt ccaaaatgct ttctttcact tcttccattc ctctgtccac 1200aaaaaaagta gtgtgttctt gagcctatat aagagagggt cacacgctcc agtcgactca 1260ccatcgatcc atctgacggt tagttccaag ggaaagaaga a 130151243DNAOryza sativamisc_featurePRO0061 - beta-expansin EXPB9 5aaaaccaccg agggacctga tctgcaccgg ttttgatagt tgagggaccc gttgtgtctg 60gttttccgat cgagggacga aaatcggatt cggtgtaaag ttaagggacc tcagatgaac 120ttattccgga gcatgattgg gaagggagga cataaggccc atgtcgcatg tgtttggacg 180gtccagatct ccagatcact cagcaggatc ggccgcgttc gcgtagcacc cgcggtttga 240ttcggcttcc cgcaaggcgg cggccggtgg ccgtgccgcc gtagcttccg ccggaagcga 300gcacgccgcc gccgccgacc cggctctgcg tttgcaccgc cttgcacgcg atacatcggg 360atagatagct actactctct ccgtttcaca atgtaaatca ttctactatt ttccacattc 420atattgatgt taatgaatat agacatatat atctatttag attcattaac atcaatatga 480atgtaggaaa tgctagaatg acttacattg tgaattgtga aatggacgaa gtacctacga 540tggatggatg caggatcatg aaagaattaa tgcaagatcg tatctgccgc atgcaaaatc 600ttactaattg cgctgcatat atgcatgaca gcctgcatgc gggcgtgtaa gcgtgttcat 660ccattaggaa gtaaccttgt cattacttat accagtacta catactatat agtattgatt 720tcatgagcaa atctacaaaa ctggaaagca ataaggaata cgggactgga aaagactcaa 780cattaatcac caaatatttc gccttctcca gcagaatata tatctctcca tcttgatcac 840tgtacacact gacagtgtac gcataaacgc agcagccagc ttaactgtcg tctcaccgtc 900gcacactggc cttccatctc aggctagctt tctcagccac ccatcgtaca tgtcaactcg 960gcgcgcgcac aggcacaaat tacgtacaaa acgcatgacc aaatcaaaac caccggagaa 1020gaatcgctcc cgcgcgcggc ggcggcgcgc acgtacgaat gcacgcacgc acgcccaacc 1080ccacgacacg atcgcgcgcg acgccggcga caccggccat ccacccgcgc cctcacctcg 1140ccgactataa atacgtaggc atctgcttga tcttgtcatc catctcacca ccaaaaaaaa 1200aggaaaaaaa aacaaaacac accaagccaa ataaaagcga caa 124361019DNAOryza sativamisc_featurePRO0063 - structural protein 6cctagctata tgcagaggtt gacaggttgt ctcttagatc gattaataat atcacattga 60tgcaattaat tatctgagat caataaagtt tttctttatg ttaaattaat atcagtaata 120gatgctaagt ccttcattag tagtatccca catttaatca cagttggaca cacaaaaaaa 180aaggcaatgc cattaatatg ccatctctct tgttttccat tgcctaccaa gtgccatatg 240atatcatcat caggcacacc aatccataac tagttcatta gagcaagttt aataatagag 300ctaactataa gcttataatt tatattggag taaacatgta tagtaaatga gctataaggt 360tatttctttt tttctcctcc tctctctatc tcttacctat atatttaatg tatttgtctt 420gaagtatgtg aatagctagc tcttgtatga gagccaatcc tctgcatttt ttaaattctc 480tttcctccac ataagcatat agttggctta tagcctgcta ttatacttgg tcttagtaca 540ctaacccccc ttacatgcaa tgcaagctgt ctaattaaaa gggtttcaca acattttgaa 600tgccactact agctcccaac cacaaccaca gatctagcta gggtttgttc atttctctcc 660tctctcctcc tcctcctttc cgttgtgcca attcatccaa agtcattgag agccatacta 720ctccatatca tattactcct acatgtgtac tacatttata ttgatgatct gtaagagcaa 780aagtattaat ggggatcaca ggattgcagt aacagcagca ggtaccccct cctttaacat 840ccgcagttac gcctcccacc taccgtcttc tctgccgatc gatgacgatg agcttctcct 900ccgctataaa tcctctcccc tcctctctcc ctcctcctcc aactccacat cgatcagcag 960cagcagcagc ttgcacactc gagcttagct tagcttttgc aagagagatc gagctagag 101971212DNAOryza sativamisc_featurePRO0081 - putative caffeoyl-CoA 3-O-methyltransferase 7atggtgccat gtcaataaga catcataata gaaactacac tccacaaccc atagtttctt 60aaagtgggtc attaataaat acatcatcta tcttttctat caatcatatt tattctttat 120ctattatgac ggcactattt tctcccaatg taaaacttga taatgtctag tgcataggtt 180ctcgtgttga agctgtttct tacatgagac ccagtttctt cttctctcca ctctctctta 240attaatataa tgtcacataa gttaaaagtt ctagtaaata ataatatagt taatgacata 300gacaacatcc tagatgtagg gttaggagtc ttcggacagt agcaaccctg ttttgactcc 360ttttttggct gcccatccac agtcgccacc agaaaattca ctgtgcccaa atcaatggaa 420gcgcctacta gatccatcca tcttcgtgac agctccgagc tttctcctgg ttatttttct 480cccaaaaata cattcagaac acgatctcaa atttaaacta atggagtgct actgcatttc 540ttaattataa gtcgcagcac cactcattaa tcatttccat cacaggtaaa tcgtggtgag 600ctggtggttg ctactgtact actagtacta cctgtcgcag ctttgtagaa gccgttttcg 660ctgaagcttc ttcttcttcc ctgggcaaaa taattttaag caggcggaat aatattggga 720taaacagggt ggacaaaagc gtgcgatccc tttctttaac caaaccacga cgaaagcagg 780ttaggtcgcg gcaggtggtg gtggtaggaa gaagaagaaa gagaggggaa aaaaaacaaa 840aatttcacat gcatcatgca tgaagtagta catgtagtac tgagtactgt aataatgttc 900agtttactgg accgtctcaa cgggaagacc aaattaacgc ttataaaata cccttttttt 960gggcactgat catggccact acgtttggtg gctcaacaac caggtcaccg tgcgatcgat 1020cgattgctaa tttatttttt gaaaaggaag ggaggaaaaa agaccgggtg tttggtggcg 1080ccaccaaccc tgctctcgtg agccgataaa tattgctcgc cggagctctc ggttgacgac 1140ccaaccaatc gactcgcacc accaccagca gctcaagcag caacagctca aacggaggaa 1200gatctcatcg cc 121281052DNAOryza sativamisc_featurePRO0091 - prolamine RP5 8gtttttctat gaaccggtca ttaaaccgtc cccggttaga ccgaacaagc cacaataatc 60ttgaaatggg ccttgatgtg gcccaattgg tctgcctaga gcgttttggt tggcaaaaat 120caatctccta ttctcggcac gtgtgatata caatggtaag tgagatatac aattctcggc 180acggctacat tacaaggtgt cgcattgtgt caatgtttgg ttaatttgct agattcacat 240aatacatgcc aggaagttca gaacaatgtg ttgcctttca ccggaaaact ttgttggagc 300aaatgccttc ttcttttttg cttctgcttc ttgagtccat gtggaggaag cagtagatag 360ctgatgatat caggattcct tctgtgtctg tgtaggtgta gcaacaccac tataattttt 420atttagcaac acaatatcaa tttggtctat aaaagtatga attaaatcaa tccccaacca 480caattagagt aagttggtga gttattgtaa agctctgcaa agttaattta aaagttattg 540cattaactta tttcgtatca caaacaagtt ttcacaagag tattaatgga acaatgaaaa 600ccattgaaca tactataatt ttttttctta ctgaaattat ataattcaaa gagcataaac 660ccacacagtc gtaaagttcc acgtgtagtg cattatcaaa ataatagctt acaaaacata 720acaaacttag tttcaaaagt tgcaatcctt atcacattga cacataaagt gagcgatgag 780tcatgtcatt atttttttgc tcaccatcat gtatatatga tgggcataaa agttactttg 840atgatgatat caaagaacat ttttaggtgc acctaacaga atatccaaat aatatgactc 900acttagatcc taatatagca tcaagcaaaa ctaacactct aaagcaaccg atagggaaac 960atctataaat agacaagcat aatgaaaacc ctcctcatcc ttcacacaat tcaaacatta 1020tagttgaagc atagtagtag aatcctacaa aa 105291216DNAOryza sativamisc_featurePRO0095 - putative methionine aminopeptidase 9cctgatggat gatgaatcac tgatcgattt ctagttctta ttctctgaag atgaaccgaa 60gatccaagat tggtccatga aattatcctt tcttgatttg gccctccgag aatagattcc 120tgtgcaatct agtcagtagt tgttcaggtc atgtaaacgt acggtaagaa atttatgtgc 180agagggtttt ccagtttatc ctatgcattt gacctctggt catgtattga ttctgagaca 240aagtgtagtg atcgcttgat gatactagta cacattgctg ccttcttttt tgtcctgtaa 300aagatttatt attggcagca atggatggta gagagggcaa tctgcttctt agttttgagt 360ataaagtttt aagttttgag cagagtttcg aaaatttgca gtagaaagtt tgaaatttca 420aattggaagt acagtttttc aaatttccag tataaatttt taaacccact gagaaaccaa 480gagcatatgg gcgatcaaaa atttcttttc taaaggaaaa atatttttta aaaaacactt 540agtagtatat caaaattctg aggtaagctc attaggccca ttcactgtac ggcccatgaa 600gcccagtctg gtgagatggg cctacccgtg caggcagaga tggatgggcc tttaattgta 660ggcccatgtt ggaaagccca ccaaagccca ataatatatc ctcctcacct tcaaccctaa 720tcctcctctt cttctagaag actgaaaatt cctctccttt cttctctcgc cctcaccgct 780cgccgaggtt gccgtctcct tgtctcctcc gctccttgcg ccgccgccgc gacgagtcgc 840ggggaggggc ggcgatctcc atctccatct gaggcgagga gagcagggga ggtgagggga 900tcctggtgag gtgagcatcc acgtcctctt tctttttttc tgattcatct ctctctctct 960cgcacatcgg gactggaatt tgcttgcgtt cgttcgttaa gttaacccta gcttctcttc 1020tagatctgga agaaactctt cttcttttaa tttcagagcc ttaaccttaa tagtacaagt 1080aacagtttgt ttgttccccg aaaagtttgg atgccttcca aatagagaca catgttattt 1140attttggaat gtaatttgtc cctggattta ttcattcagg tttgtgatta ctggacaata 1200gaaatattta cacaat 1216101237DNAOryza sativamisc_featurePRO0111 - uclacyanin 3-like protein 10tcgttaagtt tgatgatttc tgatgaccca tggtcaccta gcggctagca gtaccatgca 60tgatcaccct ccacaaagaa atggtacagt acatctccgt cccaaaataa gtgcagccat 120gtatatccat gcctaacgtt tgaccgtccg tcttatttaa aaaaattatg aaaaatttaa 180aaatatttag tcacacataa agtattattc atgttttatc atctaatagc aacaaaaaat 240actaatcata aaattttttt taataagata aacggttaaa cgttgaacgt gaatagtgca 300aaacttattt tagaacggag ggagtacgaa gtaactccgg aactacatat agggcaatta 360ttgccctatg tatgcatata gtcaatcaat taactgctga caatggaaaa gctaatcaat 420caatcaatgg tttgattaat caaattaagc caggtcagtc cgtcagtgta cattcactaa 480ttaaattaac aggtttgttc aacggttcaa ccaacatctg ccatcaacat cttttcgttg 540cacctttctt gactctttat gctattttgc taaaaaaaaa cttctcttta catcacttat 600aacaatatat atttctgctt taatttgtaa tctttttttt ctgcgttgca acggaaatca 660cgagcgatat atggtgaaga ctgatgataa tcgtatttct gatgacccat gattccgcgg 720tgtaccatct gttctgtcaa ctaaaaagtg gagtagttcc ttgacggaag aagggagcaa 780aatagaagat attctcagtt gatctgcagt tgttgttagg tcactatatt cagaaatcgc 840agttgctgtt gtttaaattg tgtgtgacag cagacagcta attatcagta cacgtatatg 900agcaatacta gtgaatctgt actaatttaa cgagagtatt ttctatatac aaatacaaca 960gcaaaactgt gccactggcg ccgaatacgt acggacagag ctcaggcaat caggggagca 1020gcaaaagagg agagagttgg tgccaagcac aactaaaccc aactgcaccc aaaaactaat 1080cagcatttca gttcgcttta gttagtacta ccacctgcat ctctttacca acactatata 1140acccgcagtg gacctgcagt catctcacta attcagtgaa gccaccagta ctagtacggc 1200tctaatcagt tcgcgtttgc taattaactc tgccatc 1237111100DNAOryza sativamisc_featurePRO0116 - 26S proteasome regulatory particle non-ATPase subunit 11 11ctaagggcag cagccattgg gctctatagg tgtggttgca agtgcactta caagcgagca 60acctggtaga atatccccga gatcagtagt taccgtgatt ggttcagact tgagaggcta 120attttttcgt acctgtagct ttattacatc gcatttcctc ttattgaagt ttagccgagg 180tggtgcggat ggatattcag tctaacagac tcaatgaacg ctttgttgta tgacttgtac 240agtactggct gctcgaacag gatggttcag cttccagaaa tttggcaacg ctccatttca 300aagaaaatca ttcagtattt gccttcttgt tgttacattg atctcatata aagtcacttt 360gatcgttgac atcttgtttt ttggttcgtt tgccatggta gtttcccttg ctgctgggag 420gattgccgcc tgaacttttt cttttttgcg aggatgttat ttttgccaga caagaacggg 480aataagcaaa ttgtttggtg gaactaaagt aaactcgatc tctttccgag aagtgtatta 540ttttcacgtg taccatcaat ttttttgaaa gtaaatattt ttccccttta actaatgttc 600actttggacc ggataatctt acctttattt aactttgggc tatctaactc tcttctaaag 660catataaacg atcttgagta catcgattcc tacttatcat ttaactctcg tagcttaatg 720taagattatt tctttgaaat atgataaatt ggatgcatat gaatgaaaga gtcaaggatt 780aagtgattcc tcaaaaaaaa aaaagagtga aatttattta tttttcccct ttcgacacga 840agaagggctt ggttggagga aaatggccca gattcagatg accgaggccg agtaccatgg 900ggcccacaag aataataagc cccgagccca aacgctaagg cccacgagaa gccgtgcgct 960ggaagaaaga aagaaaccgc ggccgtcttc acaccgaagc ggcggacgag acgactcgca 1020gtcgcagcct ctttcctcct ccgtctctct ctcccctctt cctctcctcc gcgcggcgaa 1080cgaagcgagc gagcggcggc 1100121216DNAOryza sativamisc_featurePRO0117 - putative 40S ribosomal protein 12cgtgttcatg ttcgcattta ggattggact tttttaggat ggagaggata tgtcctaacg 60gaaatgtcat gtctatgctc cgatcttata aatttgttca atagcgttgc aaacgcgatc 120attaaaaagg cggtaagaga actaccacat tttcgaaagc ccattctctt cgtgagttac 180tggaattatt tggcatagca catgcataaa gatgctttag taatgagctc aataaaacac 240gacagctttg catgtagcca caatgctata gtaaatgagt tgtacttctt ttgcattgca 300aagtggtact gaccttgttt aggcagctag cttcattcat tttttgaatt ctatagttat 360agttataaag attatcataa tttagataag aatccggtat gtttgagaag ctggagtttc 420tagagaagct ataacaactc gaagctccct aaacagagcc attgaacatt gagctgtcca 480gtatatcatg acaaaatgat acattttgca tgggcatatg tgtctaagaa aacaaacatc 540acaattcaat gagtcactct aaaaaaaaag gcaaaacact caacaaaacc ataccgtgaa 600agtgaaccta taatgaaatg aaattttgat aagcatgctt acccaggtgg aaatttcaat 660ctaagaacaa tttccaaaac caccgtccat agaaatatgt ggaattcatt cagaattttc 720ataccacacg ataaaattta tagggaattt aacttttgcc atttttaccg aacaccacct 780tttcatttgc tcctataatg ttatcgaaaa gagagtgttt gttaattatt tgtcactttt 840atcacgacat gtagccgtga caacgtggcg ttcctcgtgg agcccacccg tcagccgccg 900tacgcaccac catcaaagaa ttcaagacgg agagcgtcgt cgccgtcggc aaggcggcgt 960gttttgttca ctgtacgttg cttcggcgtg ggcccaatct tgttcgggcc
taactagttc 1020ttcccagccc aggcccatta agcctaccaa cccggacggc ccgggaggag ctagggtttc 1080acccttcact atataaacct ctctctcctc ctccggccgc cgcctccgaa gccctagctc 1140ctcccgccgc cgccgccgcc gccgccgccg cctctccact cgagagaccc agccgccgcc 1200gccgccgccg ccgcca 1216131210DNAOryza sativamisc_featurePRO0122 - chlorophyll a/b-binding protein presursor (Cab27) 13cagatgccac agtatggtgt accaccagct gctccacacc atgctccacc ggctggccaa 60ccaatgtatt tcccgaaata atctatcttt atccgatgta caagcaatta gagcaattgc 120aaatgttgcc tgcaatactc gggtctgggt atcttctctt caaattttgg gttgtaactc 180gtctatgcag ctattcatat tgtaactcag tgagctccct gtcgcaaatg tgcctctgcg 240tcagtcgctg tctgtaaact gtccggcaat tagaaattcc catccttagc atgcctggta 300ttgttcagct cgaaactgaa atttttcttc gtgccctata ttttttcggt gtagataagt 360gttccgctgg aattttatgc aggtgctgta ccctatgtgc tgcttttttt ttgtgtgggg 420cgcccccccg gggggggggg ggggtttcct ggcatgattg caaataagaa ccccggggca 480aatctgctgg ttggttgcaa ataataaccc ctccaaatct gcgcagatga aaccccattc 540aggacatgaa ttacgattgt tcatgagcta tttggatcat ggaaagattg gaaacaaaca 600cttacgtcaa ggtttctact aattacgtga ttccgatttc agagtcagcc atggctatac 660tgcctttgct ccagtaaaca tcgctgctct agtaacaaac attgcagtaa acatcacaac 720tatccaattc ccttgttgct gctctagtaa aaaacattgc aattatccaa ttcccagata 780ttttctttca ctactccaaa acctaaagta catatacgtg agttgagtga tccagcaaca 840taaaaatccg aggctccgag cgatctgcac caaccatctc acccgtccga cgtggcagca 900gcaaccagcc acagctgaga cctccatcca atagaaaccc tccctttgat tcccccgtat 960cccggcatcc ggataacgct ggataagagg cgacgcctcc cattggccac acccacccaa 1020caacgcatcc tggccgtccg atccaccccc accgccgatc tccgccgtcc gtcgccgccc 1080tcgccaccgt ggccacctgg cagcgccggc cactcccgga cagtttaata caagccacgc 1140ctttgctccg tgccggccaa aacgtaccct tgtgactaca cccgcttcgc ttcctcccct 1200ctctaagccg 1210141179DNAOryza sativamisc_featurePRO0123 - putative protochlorophyllide reductase 14ttgcagttgt gaccaagtaa gctgagcatg cccttaactt cacctagaaa aaagtatact 60tggcttaact gctagtaaga catttcagaa ctgagactgg tgtacgcatt tcatgcaagc 120cattaccact ttacctgaca ttttggacag agattagaaa tagtttcgta ctacctgcaa 180gttgcaactt gaaaagtgaa atttgttcct tgctaatata ttggcgtgta attcttttat 240gcgttagcgt aaaaagttga aatttgggtc aagttactgg tcagattaac cagtaactgg 300ttaaagttga aagatggtct tttagtaatg gagggagtac tacactatcc tcagctgatt 360taaatcttat tccgtcggtg gtgatttcgt caatctccca acttagtttt tcaatatatt 420cataggatag agtgtgcata tgtgtgttta tagggatgag tctacgcgcc ttatgaacac 480ctacttttgt actgtatttg tcaatgaaaa gaaaatctta ccaatgctgc gatgctgaca 540ccaagaagag gcgatgaaaa gtgcaacgga tatcgtgcca cgtcggttgc caagtcagca 600cagacccaat gggcctttcc tacgtgtctc ggccacagcc agtcgtttac cgcacgttca 660catgggcacg aactcgcgtc atcttcccac gcaaaacgac agatctgccc tatctggtcc 720cacccatcag tggcccacac ctcccatgct gcattatttg cgactcccat cccgtcctcc 780acgcccaaac accgcacacg ggtcgcgata gccacgaccc aatcacacaa cgccacgtca 840ccatatgtta cgggcagcca tgcgcagaag atcccgcgac gtcgctgtcc cccgtgtcgg 900ttacgaaaaa atatcccacc acgtgtcgct ttcacaggac aatatctcga aggaaaaaaa 960tcgtagcgga aaatccgagg cacgagctgc gattggctgg gaggcgtcca gcgtggtggg 1020gggcccaccc ccttatcctt agcccgtggc gctcctcgct cctcgggtcc gtgtataaat 1080accctccgga actcactctt gctggtcacc aacacgaagc aaaaggacac cagaaacata 1140gtacacttga gctcactcca aactcaaaca ctcacacca 1179151808DNAOryza sativamisc_featurePRO0133 - chitinase Cht-3 15tttggcgcgg ggcagaagag tggactttaa ctttcttttt aataaaatct ccaattaata 60tgtaattata atatactttt aatcaaaaca tgcaaagcta gcagtattta catcactaga 120agtaaatctt tcttgctcat gatgcttcag ccggacggaa ccctaaaata tagatggggc 180ggatacactc gattaaaaca gctaattgca acacatatca tataaggttt tggaattcat 240accaaatgct ccgaaattcg tctatttcga tgaggcccaa gacatgacct cctgtttcgc 300ccatagttta tggtgtttgg taaaatttgg ttaaaatctg tctattttag taggtcccga 360aattcttatg caattgaatc ctagaaccct atcatattta tattgcaatt gcacaaaaat 420aatgtgcaat caatatattc caattgcaat acatatcaag catgaggtgt aatacatatc 480cagccgctag cactgggtct gttgaggtgc ttcttgcagc aacagctgca atctgtttgg 540ctaggctgtt ggcgccaggc actgctgtcg tgctgcaaca atggcacatt cgtcgagcac 600acaaccgcgc ctatgcacag cgcaagctcg ctgccttgga ccgtggttcc agtgttgcat 660caaggcttag tggattgagc gagaagacga actgacaatg ccaaagatgc gatgctgcga 720gtgtggactg cggaagatga atcgagatca atcaattcgt tatgcttgaa aggctggaat 780aactgatcag ttggctggat cgatggtatg tactagataa tatgcggtct aggcctagac 840caagaagcag aagaggagtc gggtcgggag tgtggggcga cgtaggctgt agctgggccg 900gccgccccag gccgcctaat gagtgtgtcc gcccctggcc tgacacgatg ggtaattaaa 960tagttatgca tgtccctctt tgtctaaaca atatgtataa aattgacgat atcttgggca 1020aaatcactgg gcatggcaca caggagagct actttagcga catgaatcta ggcgaaaatc 1080tattgaacca aaaatcgact gtaatctcat gaaaattttc gtcataatta tagcaaaatc 1140gttgttggat tgattgcacg agaaaacaga agaagggagc taggtgatat tatattgttt 1200tgttgcctac ataaatctta aagcaatcga atggtctaaa atttacaaga tttttaaaga 1260ggttttcgta ccgtatagac cccggccggg tcaaacttat ttggtcgtcg ctggttgttt 1320gtagcacgcc agctccatat atgtggattg cagctggtct atgataagtt cggtcgatct 1380gagatcaatc tatcaatcgt caaccctttg cctttgttag cgagctagcg tgtacacatt 1440tcaattatat atggtgcatg catggcatcc acgcctccac ggtcaacgtg gaaatatctc 1500tggaaactta ctttttctaa ataactgaac ggattggagg caggagacaa atttgaccaa 1560cacaatatat ccacgacggc tagacaatac tagtagatgc atgcatggaa ggatatagta 1620gtacttgtta atcgtggaaa ctttggtaat gcgaatgcat ttcaattcgt tgctgaagat 1680cgatgcacca tgcatatcca tctctatata aagccatgcg atcccaccga ttcttgcaca 1740cacactagct acttctactt ctatcatacc aaacaaacta gcttaatttg cattgcatca 1800cattgccg 1808161828DNAOryza sativamisc_featurePRO0151 WSI18 16gcttgagtca tagggagaaa acaaatcgat catatttgac tcttttccct ccatctctct 60taccggcaaa aaaagtagta ctggtttata tgtaaagtaa gattctttaa ttatgtgaga 120tccggcttaa tgcttttctt ttgtcacata tactgcattg caacaattgc catatattca 180cttctgccat cccattatat agcaactcaa gaatggattg atatatcccc tattactaat 240ctagacatgt taaggctgag ttgggcagtc catcttccca acccaccacc ttcgtttttc 300gcgcacatac ttttcaaact actaaatggt gtgtttttta aaaatatttt caatacaaaa 360gttgctttaa aaaattatat tgatccattt ttttaaaaaa aatagctaat acttaattaa 420tcacgtgtta aaagaccgct ccgttttgcg tgcaggaggg ataggttcac atcctgcatt 480accgaacaca gcctaaatct tgttgtctag attcgtagta ctggatatat taaatcatgt 540tctaagttac tatatactga gatgaataga ataagtaaaa ttagacccac cttaagtctt 600gatgaagtta ctactagctg cgtttgggag gacttcccaa aaaaaaaagt attagccatt 660agcacgtgat taattaagta ctagtttaaa aaacttaaaa aataaattaa tatgattctc 720ttaagtaact ctcctataga aaacttttac aaaattacac cgtttaatag tttggaaaat 780atgtcagtaa aaaataagag agtagaagtt atgaaagtta gaaaaagaat tgttttagta 840gtatacagtt ataaactatt ccctctgttc taaaacataa gggattatgg atggattcga 900catgtaccag taccatgaat cgaatccaga caagtttttt atgcatattt attctactat 960aatatatcac atctgctcta aatatcttat atttcgaggt ggagactgtc gctatgtttt 1020tctgcccgtt gctaagcaca cgccaccccc gatgcgggga cgcctctggc cttcttgcca 1080cgataattga atggaacttc cacattcaga ttcgataggt gaccgtcgac tccaagtgct 1140ttgcacaaaa caactccggc ctcccggcca ccagtcacac gactcacggc actaccaccc 1200ctgactccct gaggcggacc tgccactgtt ctgcatgcga agctatctaa aattctgaag 1260caaagaaagc acagcacatg ctccgggaca cgcgccaccc ggcggaaaag ggctcggtgt 1320ggcgatctca cagccgcata tcgcatttca caagccgccc atctccaccg gcttcacgag 1380gctcatcgcg gcacgaccgc gcacggaacg cacgcggccg acccgcgcgc ctcgatgcgc 1440gagcccatcc gccgcgtcct ccctttgcct ttgccgctat cctctcggtc gtatcccgtt 1500tctctgtctt ttgctccccg gcgcgcgcca gttcggagta ccagcgaaac ccggacacct 1560ggtacacctc cgccggccac aacgcgtgtc ccccctacgt ggccgcgcag cacatgccca 1620tgcgcgacac gtgcacctcc tcatccaaac tctcaagtct caacggtcct ataaatgcac 1680ggatagcctc aagctgctcg tcacaaggca agaggcaaga ggcaagagca tccgtattaa 1740ccagcctttt gagacttgag agtgtgtgtg actcgatcca gcgtagtttc agttcgtgtg 1800ttggtgagtg attccagcca agtttgcg 1828171267DNAOryza sativamisc_featurePRO0169 - aquaporine 17cgtcctcctt ttgtaacggc tcgcaaatac aatgggttgt ttagattcat gtcattttaa 60atcatattat tttttataaa gttatcaaaa tgtacatata tttatttatt tttaccaaac 120tttactaaat gagataatcc aacaaatggc atttaaagcg ttcaaatcca agaaatgcca 180tcgccgttat gcttccgtcc gtttcacgcc gttaaaatac aatgttcatc ctataacact 240taatggtgtg gaatggacgg aaccctaacg gcgatggcat ttttgggata aagtcgtttg 300tacgatggca tttcttagaa ctcatatttg tcgatggcat tttttgaatt tggatgattg 360tcaatggtat tttttggatt atctcttagt aaatacataa ggaatcatgc caaaacttga 420caatattgtc aacttatcaa aatttaattg ggattatttt ggcgataata tgaacagccc 480ttacatttct gaagaattat agctcaaata tggctatggc cctgtttgga ttcggagggc 540tatttaatag ccctccggaa tcttgctatt taagagtatt aaacgtagat tactgataaa 600actcattcca taacccctac gctattctac gagacgaatc taacgaggta tattaatcca 660tgatttgcta cagtaatcag ccgctaatcg tggattaata tacatcatta gattcgtctc 720gtaaaatagg ctagggatta tggaatcggt tttatcggta atctatgttt aatacttcta 780aatagcaaga ttccgaaggg ctatttaata gctcggagca tccaaacaag gcctatgttt 840agatccaaac ttccaacttt ttctatcaca ttaaactgtc atacatacat aacttttcag 900tcacatcgta ccaatttcaa cccaaacttt caactttgga agaactaaac acagcatatg 960acagtgcagt tcagctcaat tttgttcgga gcctaaaaaa aagaaaagaa aaaaagctca 1020atttggataa ggctatgaat aaactcaaaa aagcatccaa cctaaccacc acactggccc 1080accagggccc acgctccact cccgtgatca tcacctcctt ccctttccag aaccaccttc 1140tccttccttc ctcctcttct tcttcagtgt actctgcctt tataacaccc tactcctctc 1200tctcacctcc accatctagc tcactcacac agtctccact cacacgcatt gcagaggaga 1260ggcgaca 1267181130DNAOryza sativamisc_featurePRO0170 - High mobility group protein 18catgcggcta atgtagatgc tcactgcgct agtagtaagg tactccagta cattatggaa 60tatacaaagc tgtaatactc gtatcagcaa gagagaggca cacaagttgt agcagtagca 120caggattaga aaaacgggac gacaaatagt aatggaaaaa caaaaaaaaa caaggaaaca 180catggcaata taaatggaga aatcacaaga ggaacagaat ccgggcaata cgctgcgaaa 240gtactcgtac gtaaaaaaaa gaggcgcatt catgtgtgga cagcgtgcag cagaagcagg 300gatttgaaac cactcaaatc caccactgca aaccttcaaa cgaggccatg gtttgaagca 360tagaaagcac aggtaagaag cacaacgccc tcgctctcca ccctcccacc caatcgcgac 420gcacctcgcg gatcggtgac gtggcctcgc cccccaaaaa tatcccgcgg cgtgaagctg 480acaccccggg cccacccacc tgtcacgttg gcacatgttg gttatggttc ccggccgcac 540caaaatatca acgcggcgcg gcccaaaatt tccaaaatcc cgcccaagcc cctggcgcgt 600gccgctcttc cacccaggtc cctctcgtaa tccataatgg cgtgtgtacc ctcggctggt 660tgtacgtggg cgggttaccc tgggggtgtg ggtggatgac gggtgggccc ggaggaggtc 720cggccccgcg cgtcatcgcg gggcggggtg tagcgggtgc gaaaaggagg cgatcggtac 780gaaaattcaa attaggaggt ggggggcggg gcccttggag aataagcgga atcgcagata 840tgcccctgac ttggcttggc tcctcttctt cttatccctt gtcctcgcaa ccccgcttcc 900ttctctcctc tcctcttctc ttctcttctc tggtggtgtg ggtgtgtccc tgtctcccct 960ctccttcctc ctctcctttc ccctcctctc ttcccccctc tcacaagaga gagagcgcca 1020gactctcccc aggtgaggtg agaccagtct ttttgctcga ttcgacgcgc ctttcacgcc 1080gcctcgcgcg gatctgaccg cttccctcgc ccttctcgca ggattcagcc 1130191230DNAOryza sativamisc_featurePRO0171 - reversibly glycosylated protein RGP1 19tagtaccatt cttccctcgt gagcataaat gtattcatac aaaatagtaa aatgtatcct 60cacaaagatt gtaagtatat ctcgcaacta taaatatgtt gtcattttag taacaattgt 120tcataaaata gtaatcatgt tctccataac agtaaatgac gaggcgttaa tagtggttta 180ggttctcatg attgtaaatg ttgagtcgct tgtagcggct taagatatag tagagagtat 240atctagtttt atcaagacaa acattgcgta atgcctcgga cctaatataa aagtaggaat 300tttaaccttt gagaaactgt aaccaattga aactgcaagc tttaaaaaaa catctattgg 360aagtgatatt atatagacaa aataagtttc ttactcttac tctctcagtt tcaagttata 420aaatgttttg gctttggtca aaatcaaact tcttcaagtt taatcaagtt tatagaaaaa 480atagtaatat ccaagataaa tttattataa aaatatattt aattattatt ttaataaaac 540taatttggta atgtaaatat tactatattt gtctataaac ttagtcaaat ttaaaacagt 600ttaactttga ccaaagtcaa aacatcttat aacctgaaat ggatggagta tttgtttgtt 660tctattttag gaaacggccg tttctttcca ttgattttga gataagcaga gctttaaacc 720actgccacta ttgtgcattt catttgattt aacactttta ccccttatct ccaataaaaa 780cgatattaag atacccctat cttttatcca ccgcttggaa caaaccaaaa aaaataaaaa 840ttcaaacctt ctacactggt acacacgttc tctctttcca tgcaccgaca ggtctctccc 900agatccaacc caaaataaat ttggacgcat cccaaaattc ggcaaacata tgacgcaaac 960caaaacaaaa taggcacaaa ataatataat actcctatct aattaattat acacaatttt 1020ttttaaaaaa aaagcaaggc aagcgaagca aagcaaagaa ggaaacgaat aacaaagtcg 1080tcgtcctccc ggagctcccg ctctataaat cgctcctcct ccccacccac ccaaacccac 1140acacacctca cacctcacca ccatcacctc ctcctcctcc tcctcttcct ccgcgcgcgc 1200gagatccagg gagagggaga gggagagatc 1230201234DNAOryza sativamisc_featurePRO0173 - cytosolic MDH 20gtttggttgg tgaccgcaat ttgctatacc aaaatcttag acacagttga attaagctac 60actttattag cacattggcc cgtgcgttat attgtcattt tctagccaaa gtttgccata 120attgtggcta acaaattgtt ggccacattt tggctacgtt cgataggaca tgttcccaac 180ttctccttct cgtttttcgc gcgtacgctt tttcaaactg ttaaacggtg tgttttttgc 240aaaatatttt tttacgaaag ttgcttaaaa aattatatta atctattttt tttaaaaaaa 300gtagctaaaa cttaattaat ctcacgctag acgctgcttc gttttacgtg tcgggtaccc 360aaccctcact cccgaacaca gcctttgtgt ggtttactac agttatagta aagctagtct 420ccatccaaac aatcctttag tccatataac ttcgtatact ccaaaattcc actcgttcta 480cggacatcac taatacgaag atcaagtgga agatagatat ttttaatgac atgttatttt 540cagtgaacac ttgaggtcct cacgatccac aaacacacat tttcgtagat aagttctgaa 600atactccata cggcggttgt cacgatgtca tgatcgtcgt tacccaagga agaagaaaag 660agtggcatct tctccacgcc agtgttccca acggagcatc ttttcttccc ccacacggca 720tcgacgtcac actttctggt gcaaacttta ataattagtc caaaaacaaa aaaagaattt 780cggccacatc ttctcccgaa acgccaggtg ggccccacct gcatcactga cagcctgtcc 840ccacaacgcg cagtcgtgtc cccacctgtc aggatgttag cgtctccgtt gcaggtttcc 900cagatcccat cgccgatctg tgggccagcg cccacggtgt cacgcccgcg cacacctggc 960tccaacccac ccaccccacg cgctccgtgg ccgacagcgt ggacccacct aggtggggcc 1020caccgtcagt gggagatggg taggggagcc cccacgtggg agcaacgggg gttctccggg 1080ctccccgtcg ccgcgaggtt aaataacggc cacccgtttc cccctctctc gcaaaactca 1140cccaaaagag cagcgtcgcc tctcctcctc ccccctaacc cctacgcttc cagaaccttc 1200tcgaagctcc cgctcccccc ccccttccgc tcca 1234211553DNAOryza sativamisc_featurePRO0175 RAB21 21gtcaccaccg tcatgtacga ggctgcttca ccactgcctc actgccacca gcgtctcccg 60ccgcgtgcaa tacaagaaga aacatcgaac ggtcatataa ggtaagaccc actaccgatt 120taacctatca ttcccacaat ctaatccact tatttctctt cccatgatct tatcctctca 180tttctcctca ctacttttgc atttgtagga aacacaatga caccgtcgaa gaaagctggt 240ggagcaccgt agccagcaat caccaaaaca cagaggggag gaggtcggca gcggccatgc 300ggacggcgat gagacaacgc gacgcaaaga gggaggagga cgttggcgat catgctggtg 360ttggcggagg aggtcactgg ccatgcgaat gacagcgggg cagcgcaaca caaaaagggg 420ggaggatgcc ggcgaccacg ctagtaccat gaagcaagat gatgtgaaag ggaggaccgg 480acgagggttg gacctctgcc gccgacgtga agagcgtgat gtgtagaagg agatgttaga 540ccagatgccg acgcaactta gccctgcaag tcacccgact gcatatcgct gcttgccctc 600gtcctcatgt acacaatcag cttgcttatc tctccatact tgtcgtttgt ttcccgtggc 660cgaaatagaa gaagacagag gtgggttttg ttggagagtt ttagtggtat tgtaggccta 720tttgtaattt tgttgtactt tattgtatta atcaataaag gtgtttcatt ctattttgac 780tcaatgttga atccattgat ctcttggtgt tgcactcagt atgttagaat attcattccg 840ttgaaacaat cttggttaag ggttggaaca tttttatctg ttcggtgaaa catccgtaat 900attttcgttg aaacaatttt tatccgacag caccgtccaa caatttacac caatttggac 960gtgtgataca tagcagtccc caagtgaaac tgaccaccag ttgaaaggta tacaaagtga 1020acttattcat ctaaaagacc gcagagatgg gccgtggccg tggctgcgaa acgacagcgt 1080tcaggcccat gagccattta ttttttaaaa aaatatttca acaaaaaaga gaacggataa 1140aatccatcga aaaaaaaaaa ctttcctacg catcctctcc tatctccatc cacggcgagc 1200actcatccaa accgtccatc cacgcgcaca gtacacacac atagttatcg tctctccccc 1260cgatgagtca ccacccgtgt cttcgagaaa cgcctcgccc gacaccgtac gtgcgccacc 1320gccgcgcctg ccgcctggac acgtccggct cctctcccgc cgcgctggcc accgtccacc 1380ggctcccgca cacgtctccc tgtctccctc cacccatgcc gtggcaatcg agctcatctc 1440ctcgcctcct ccggcttata aatggcggcc accaccttca cctgcttgca caccacagca 1500agagctaagt gagctagcca ctgatcagaa gaacacctcg atctctgaga gtg 1553221087DNAOryza sativamisc_featurePRO0177 - Cdc2-1 22cagacaccta gaatatagac attcccaaaa aataatcact atgcatcagc atcactatac 60atgacttggg tctagtgatg gaagtggata gttccactac ctacataaaa acccactact 120agtttattac ttttcacatg atagcataaa atttaaagaa aaaataaaca gaagtggaat 180aagcgaaaaa ccccgcttac ccgccccatt tacatcccta cttggatcct gcatgtcagt 240aagatatcag aattatatgt tttagaatta tatgtttttt tggaaggtgg aaatcggatt 300attagacgca acataccaag tggcgtatac ttggcttcac tctttccatc agagcaagcg 360taaaagatca cgtattcacg tcacatggag taactgagcg aatttttttc atttttaaat 420ttttgttttt taatatttac ataaatatta taccggcgaa aatatttaca aaagtagacc 480ctgctgccct tctccttctc gagaagagcg gcagggtgat gtcagggaca gaaataaact 540ccaaaaatgc atttttggct gggcgaaaat tgcacttacc cccttgctgc cctctacaaa 600ggttgcaagg gacctcagtg caaaatacgc acaccttgcc gtcctccact tggacggcat 660gggctatttc tgtaaatatt ttggatggta taatatttct gtaaatatta aaaaataaaa 720atttaaaaat gaaaaaattc tatctgggct cccttctctc atctcacacg gcccaccaca 780caatcccggc ccacatattt cctgggccca tttccgtgtg aatggagacg gcccattggc 840gcgcacatgc ggaaaagcgt acacacgatt cgaaatttga aatctcaaaa agcgcccgtt 900agagcgcgtc ccctccaacg gctatcccca atacaaaaga tcactcgaat cccccccaaa 960tcgaccaaac cctaaatcca cgcgcattcc acaccaccca accagcgaga gagagatggc 1020ggcgctccac caccaggcgg cggcggcgcc ggtgacgacg acgacggacg ggggcgagct 1080gcgggcg 1087231272DNAOryza sativamisc_featureTC89946 (PRO0110) 23tttgacgact gaatcgnggc tcgcctctgc ggcggccgct ctagattagn gtttcccctg 60tctgttgtaa ttcggcacga gggctgatca agagctctta attagctagc tagtgattag 120ctgcgcttgt gatcgatcga tctcgggtac gtagcaatgg cgtccaaggc gttcgctctg 180ttcctggccg tgaacctcgt cgtgctcggg gtggcaagcg cctgcggcgg cagcccgtcg 240tgcccgacgc cgacgccgtc gaccccgaca
ccgtcaacgc cgacgccgac gccgtcggcg 300ttcgggaggt gcccccgcga cgcgctgaag ctgggcgtgt gcgccaacgt gctgggcctg 360atcaaggcca aggtgggcgt gcctccggcg gagccgtgct gcccgctgct ggaggggctc 420gtcgacctcg aggcggcggt gtgcctctgc acggccatca ggggcaacat cctcggaatc 480aacctcaacc tccccatcga cctcagcctc atcctcaact actgcggcaa gaccgtcccc 540accggcttca agtgctaagc agcgtgcata tgcaatgcct gcatgggttg atcctacgta 600cggtgattag ttggctttga cgactcttga tttgatttgc ttgctgctct gtttatttgc 660tactacgtta cgtacgtact ttgcatgcaa cgcaacgcat gatcgatcgt gcatgctggc 720tgtttgtacg tatcacggta ccagtttgga ttctctctgt actctctcct ttgtcttctt 780tgtagtactc ttattcccgc tatccgtacg tgcgcatttg ttgtaagggc cggtgctagc 840ttgtgtgccg gtaccaactt ctaataaagc tatgggtgga acttcaaaaa aaataaaaaa 900aaaactggag ggggggcccg ggtccaattt agactataat gagtttaaca ccccgctcat 960cggccgaaga taacaacacc gggcttggaa aacctagact gcccaactaa tggacggaag 1020acagactctt ggactgaaac tgaacgaaac aagaccaccc accccatcta accacagcca 1080cctaccgcca aagattccaa taatgtgaat cagtcggtaa tagaacactc ctcttgtacg 1140attttactgc ccgcgccacc cctcggtacg cacttatata tatcgggccg tagtaatttc 1200ctggttccgt cacttccctc atcgcacctg ctagtcgtgg cttacatacg tgcgtcctct 1260tattatcgag cg 1272242425DNAOryza sativamisc_featureTC90358 (PRO0005) 24cccacattga ataattattt taaataattt aagttttttt tttttggctt tagatatatt 60cccaatcccc aacctcccaa taatccgatc tctcccagtt ctgttcggat caaggctgtg 120tcgatcgcaa aaaagaaaaa aaaaacaatt tccttttggg gtggttcatc tgttgatcac 180ttctttgttt cccgcgtttt gttggggatt cgattttcgg gttaagattt tctacacgat 240ggccttgaac ttggctcaga gcgccgcggc ggcagcgtgc ttcgcgaccg ccggtgatgc 300gcggcgagct gcttcggtgg tcgccatgcc gtcgtcgtcg tcgtcggcca cgacgagcct 360gaggatgaag aggcaggcgg cgtgcgagcc ggtggcgtgc cgggcggtgg ccaggcacgt 420ggcggcggcg gcggcgagca gcaggaggaa cggcgtgccg gtgttcgtga tgatgccgct 480ggacacggtg agcaagtgcg ggagcgcgct gaaccggagg aaggcggtgg cggcgagcct 540ggcggcgctg aagagcgccg gcgtggaggg gatcatggtg gacgtgtggt ggggcatcgt 600ggagagcgag ggccccggcc ggtacaactt cgacggctac gtggagctca tggagatggc 660ccgcaagacc ggcctcaagg tccaggccgt catgtccttc caccagtgcg gcggcaacgt 720cggcgactcc gtcaacatcc cgctcccgag gtgggtggtg gaggagatgg agaaggacaa 780cgacctcgcc tacaccgacc aatggggacg ccgcaacttc gagtacatct ccctcggctg 840cgacgccatg cccgtcttca agggccgcac gcccgtcgag tgctacaccg acttcatgcg 900cgccttccgc gaccacttcg cctccttcct cggcgacacc atcgtcgaaa tccaagtcgg 960catgggcccc gccggcgagc ttcggtaccc gtcctacccg gagagcaacg gcacctggag 1020gttccccggc atcggcgcct tccaatgcaa cgacaggtac atgcgtagca gcctgaaggc 1080ggcggcggag gcgaggggca agccggtagt ggggccacgg cgggccgacg gacgccggcg 1140gctacaacaa ctggccggaa gacacggtgt tcttccgcgg cgactgcggc gggtggagca 1200ccgagtacgg cgagttcttc ctgtcgtggt attcgcagat gctgctggag cacggcgagc 1260gcgtgctgtc gggcgcgacg tccgtgttcg gcgacggcgc cggcgccaag atctcggtca 1320aggtggccgg catccactgg cactacggca cgcggtcgca cgcgccggag ctcacggcgg 1380ggtactacaa cacgcggcac cgcgagcggc tacctcccga tcgcgcgcat gctggcgcgc 1440cacggcgccg tgctcaactt cacctgcgtg gagatgcgcg accacgagca gccgcaggag 1500gcgcagtgca tgcccgaggc gctcgtcagg caggtggccg ccgcggcgcg cgcggcgnga 1560cgtcgggctc gccggggaga acgcgctgcc gcggtacgac ggcacggcgc acgaccaggt 1620ggtcgccgcc gccgccgacc gcgcggcgaa ggaccggatg gtcgccttca cctacctccg 1680gatggggccc gacctcttcc acccggacaa ctggcgccgg ttcgtcgcct tcgtccgccg 1740catgtccgag tccggctcgc cgcgggaggc cgccgagagc gccgcgcacg gcgtcgcgca 1800ggccaccggc tcgctcgtgc acgaggccgc ggtcgcgctc cggagctagc accggtcaga 1860cgctcatata caccgtcgcc tcgaggtcgg attccgatgt gggatcattc gatctccctt 1920ttttttttct tctttttgcc attttgtaca gccttttggg gagctttgga tttgtgcttt 1980ttgtctcggg aggaaaaccg ctctggaggt cgaagagagc gtcattttcc tcccgttgaa 2040gatcacgaat catttacgtt agagatgatg taattaagca gggaggggag gggaacacac 2100acacactggc actcaaaagt tgttgtcacg cttggggaat atatccattt ccagccaaaa 2160aaaaaacgca gaaatgcgtt gtgttcttgc gctctggttc gttgctgctg tgggtcagat 2220tcagctggtg aaaaaactac agtactactg aaactgaaac tactagagcc tagagggaga 2280ttaagctaag ttaattgcac gagtaattac tccacggttg tgtttagggt ctacgtcggc 2340agattttgct ttctggtaga tccctaacct tatgtttgtt gggaatttta taaaggagct 2400aagtttgcct attgatttgc aatct 2425253410DNAOryza sativamisc_featureTC83635 (PRO0009) 25ccatggacac cgcctccgtc accggtggcg agcacaaggg gaaggagaag acgtgccggg 60tgtgcggcga ggaggtggcg gcgagggagg acgggaagcc gttcgtggcg tgcgccgagt 120gcggcttccc ggtgtgcaag ccctgctacg agtacgagcg cagcgagggc acccagtgct 180gcccccagtg caacacccgc tacaagcgcc acaaagggtg cccacgggtg gaaggcgacg 240aggacgacgg cggcgacatg gacgacttcg aggaggagtt ccagatcaag agccccacca 300agcagaaacc cccccacgag cccgtcaact tcgacgtcta ctcggagaac ggcgagcagc 360cggcacagaa gtggcgccct ggaggcccgg cgctctcttc cttcaccgga agcgtggctg 420ggaaggatct ggagcaggag agggagatgg agggtggcat ggagtggaag gacaggatcg 480acaagtggaa gacgaagcag gagaagcggg gcaagctcaa ccgcgacgac agcgacgacg 540acgacgacaa gaacgacgac gagtacatgc tgctcgcgga ggcgaggcag ccgctgtgga 600ggaaggtgcc gatcccgtcg agcaagatca acccgtaccg gatcgtgatc gtgctccggc 660tggtggtgct ctgcttcttc ctcaagttcc ggatcacgac gccggcgatg gacgcggtgc 720cgctgtggct ggcctcggtg atctgcgagc tgtggttcgc gctgtcgtgg atcctcgacc 780agctgcccaa gtggtcgccg gtgacgaggg agacgtacct ggaccggctg gccctccggt 840acgagcgcga cggcgagccg tgccgcctgg ccccgatcga tttcttcgtc agcacggtgg 900acccgctcaa ggagccgccc atcatcaccg ccaacaccgt gctgtccatc ctcgccgtcg 960actaccccgt cgaccgcgtc tcctgctacg tctccgacga cggcgcgtcc atgctgctct 1020tcgacacgct ctccgagacc gccgagttcg cccgccggtg ggtccccttc tgcaagaagt 1080tcaccatcga gccccgcgcc cccgagttct acttctccca gaagatcgac tacctcaagg 1140acaaggtcca gcccaccttc gtcaaagaac gccgcgccat gaagagagag tatgaggagt 1200tcaaggtgag gataaacgcg ctggtggcga aggcgcagaa gaagccggag gaagggtggg 1260tgatgcagga cgggacgcca tggccgggga acaacacgag ggaccacccg gggatgatcc 1320aggtgtacct gggcagccag ggcgcgctcg acgtcgaggg cagcgagctg ccgcggctgg 1380tgtacgtgtc ccgcgagaag cggcccggct acaaccacca caagaaggcc ggcgccatga 1440actccctcgt tcgcgtctcc gccgtgctta ccaacgcccc cttcatcctc aacctcgact 1500gcgaccacta cgtcaacaac agcaaggccg tccgcgaggc catgtgcttc ctcatggaca 1560agcagctcgg caagaagctg tgctacgtcc agttccccca gcgcttcgac ggcatcgacc 1620gccacgatcg ctacgccaac cgcaacaccg tcttcttcga catcaacatg aaggggctgg 1680acgggataca ggggccggtg tacgtgggga cggggacggt gttcaacagg caggcgctgt 1740acggatacga cccgccgcgg ccggagaaga ggccgaagat gacgtgcgac tgctggccgt 1800cgtggtgctg ctgctgctgc tgcttcggcg gggggaagcg cggcaagtcg cacaagaaca 1860agaagggcgg cggcggcggc gagggcggcg gcctcgacga gccgcgccgc gggctgctcg 1920ggttctacaa gaagaggagc aagaaggaca agctcggcgg cggcgcggcg tcgctcgccg 1980gagggaagaa agggtaccgg aagcaccagc gcgggttcga gctggaggag atcgaggagg 2040gcctcgaggg gtacgacgag ctggagcgct cgtcgctcat gtcgcagaag agcttcgaga 2100agcggttcgg ccagtcgccg gtgttcatcg cctccaccct cgtcgaggac ggcggcctcc 2160cccagggcgc cgccgccgac cccgccgccc tcatcaagga ggccatccac gtcatcagct 2220gcggctacga ggagaagacc gagtggggca aggagattgg gtggatctac gggtcggtga 2280cggaggacat cttaacgggg ttcaagatgc attgccgtgg gtggaagtcg gtgtactgca 2340cgccggcgag ggcggcattc aaggggtcgg cgcccatcaa cctgtcggat cgtctgcacc 2400aggtgctccg gtgggcgctc ggctccgtcg agatcttcat gagccgccat tgcccgctct 2460ggtaccctat ggcggccgcc tcaagtggct cgagcgcttc gcctacacca acaccatcgt 2520ctaccccttc acctccattc ccctcctcgc ctactgcacc atccccgccg tctgcctcct 2580caccggcaag ttcatcatcc ccacgcttaa caatttggcg agcatatggt tcatagcgct 2640tttcctgtcg atcatcgcga cgggggtgct ggagctgcgg tggagcgggg tgagcatcga 2700ggactggtgg aggaacgagc agttctgggt gatcggcggc gtgtcggcgc acctgttcgc 2760cgtgttccaa ggcctcctca aggtgctcgg cggcgtggac accaacttca cggtgacgtc 2820caaagccgcc gccgacgaag accgacgcgt tcggcgagct ctaactgttc aagtggacga 2880cgctgctggt gccgccgacg acgctgatca tcatcaacat ggtggggatc gtcgccggcg 2940tgtcggacgc cgtgaacaac gggtacgggt cgtggggccc gctgttcggg aagctcttct 3000tctccttctg ggtcatcctc cacctctacc ccttcctcaa ggggctcatg gggaggcaga 3060accggacgcc cacaattgtc gtgctctggt ccaacctcct cgcctccatc ttctccctcg 3120tctgggtcag gatcgacccc ttcatcccca agcccaaggg ccccgtcctc aagccatgcg 3180gggtctcgtg ctgagctgct gctgctactt ctctgtgtct ctgcattttg caagagggat 3240gaccggatgg atgattcttg ttgtatggag tattttgact tgttcatgta caagtttttg 3300tgagtgggat aaaagtgttt tgggggtaaa atttgtaaga actgaggtgg agattatact 3360cgaatttaag aacaattgtt tttgaatttt cttttaagat ttttgggagt 341026602DNAOryza sativamisc_featureTC83117 (PRO0058) 26cccccccctc gaggttcgac ccactcgtcc gctgacggtt agttccaagg gaaagaagaa 60atggaggctt cacgcaaggt gttctcggcc atgcttctca tggtgctgct gcttgcagcc 120actggtgaga tgggcgggcc ggtgatggtg gcggaggctc ggacgtgcga gtcgcagagc 180caccggttca agggcccgtg cgcccgcaag gcgaactgcg ccagcgtatg caacacggag 240ggcttccccg acggctactg ccacggcgtc cgccgccgct gcatgtgcac caagccctgc 300ccctgatcga tgaaccagca gctagcgcag cagcttgtgc cgccacctcg cgcatgtgtc 360atcgtgtcga tcgatcggat cctagctgcc ctatgaatga ataaaagtgt gtggcttatg 420cgtggttttc tcttggagaa ctttggcttt tgtggtgtta agttcgatcg ttttgtgcat 480ccaccatcca tccatcctcc cattctgctt gttctaaggt tatactacta cttgagaagg 540tgatgcaatt gtgctcaaca gtttattaat acttcatccg ttttaaaatg tttgaccccg 600tt 602271170DNAOryza sativamisc_featureTC89913 (PRO0061) 27aattcggcac gagannaaaa ggaaaaaaaa acaaaacaca ccaagccaaa taaaagcgac 60aatgggatcg ctcaccacca acatcgtcct cgccgtcgcc gtggtggcag cgctggtcgg 120cggcgggtcg tgcggcccgc ccaaggtgcc acccggcccg aacatcacga ccaactacaa 180cgccccgtgg ctccccgcca gggccacctg gtacggccag ccctacggct ccggctccac 240cgacaatggt ggcgcgtgcg ggatcaagaa cgtcaacctg cctccctaca acggcatgat 300ctcctgcggc aacgtcccaa tcttcaagga cggcagggga tgcggctcat gctacgaggt 360gaagtgtgag cagccggcgg cgtgctcgaa gcagccggtg acggtgttca tcacggacat 420gaactacgag cccatctcgg cgtaccactt cgacttctcc ggcaaggcgt tcggcgccat 480ggcttgcccg gggaaggaga ccgagctccg caaggccggc atcatcgaca tgcagttcag 540gagggtgcgc tgcaagtacc ccggcggcca gaaggtcacc ttccacgtcg agaagggctc 600caaccccaac tacctcgccg tgctcgtcaa gttcgtcgcc gacgacggtg acgtcatcca 660gatggacctc caggaggccg gattgccagc gtggaggccc atgaagctgt cgtggggcgc 720catctggagg atggacaccg ccacgccact caaggcaccc ttctccattc gcgtcaccac 780cgagtccggc aagagcctca tcgccaaaga cgtcatcccg gtcaactgga tgccagacgc 840catctacgta tcaaacgtcc agttctattg agatcggacg gaaacgatcc tcctaattta 900tttccctatt aatttgttca aatggtttcc ttctataacc tatatttttc ccgttgttag 960aaatggttcc atttcctcct acagcttact ttaagatagt tgcgcttgta tatctgcgcc 1020atcttgtaag ttgtaagatg ctgaagaaca ctatgaattc tgagcatctg attctccggg 1080aagatttact atgataaaca acagtttgat ttactatgtg tgtccccttg tttattgtat 1140gctatcctaa tacttatgaa angttttgat 117028861DNAOryza sativamisc_featureTC89985 28ccacgcgtcc gcccacgcgt ccgcgatcag cagcagcagc agcttgcaca ctcgagctta 60gcttagcttt tgcaagagag atcgagctag agatggagaa gtcgagcaag atgatggcgg 120tggcggcggt gctggtgctc gcggtggtcg gcgcggcgga ggcgaggaac atcaaggcgg 180cggcggcggc ggcggcggag agcaaggaca cggtggtgca gccgacgacg ttcccgccgt 240tcgaccgctt cgggagcgcg gtgccggcgt tcggcggcat gcccggcagc agcatcccgg 300ggttcagcct ccccggcagc agcggctcca cccccggcgg cctcggcggc ttcggcagca 360tgcccatgtt cggcggcctc ggcggcggct cacctggcct cggcggcggc atgcccggct 420cccccgccgc cgccgacaag caggccaaga agccatgaga gacctcgccg tcgccggcgg 480cgtcgccgct gctgcgcggg taatgtgctc tatgtagcgc acggcgttgc atgcaatatg 540gatggctata tgacgcgcgc gcgttatatc ttcatatgtg cagttagctt gcactgtgtc 600tagctagcgt tctattatga gtagtgtctc ttctatctct tttctttaca tgcatttgga 660ggaggattat tctatctgtt tgttggttgg ttgtgtttgt ttgttttaat taggtccctt 720cttatatttt gtgttttaat taagttcgtg atcatgtagt agtactacca ctgtttcgag 780ctcgaggcat gaataatgct aaatgtgatc attattgtgt tattgtatgg tgatggctat 840atatattact atctctgctt c 861291252DNAOryza sativamisc_featureTC89891 (PRO0081) 29cccangcgtc cgaaccaatc gactcgcacc accaccagca gctcaagcag caacagctca 60aacggaggaa gatctcatcg ccatgacgac cggcaatggc gacgcaccgg tgatcaagaa 120cgcccacagc gacatcgaca gcaccaacaa gacgctgctc aagagcgacg ccctgtacaa 180gtatgtcctg gacacgacgg tgctgccacg ggagccggag tgcatgcgcg atctgcgcct 240catcacggac aagcaccagt gggggttcat gcagtcgtcg gcggatgagg cgcagtgctg 300gggatgctgc tgaagatggc cggagcgaag aggacaatcg aggtgggtgt cttcaccggc 360tactcgctgc tggcgacggc gctggcgctg ccggaggacg ggaaggtggt ggcgatcgac 420ccggacaggg agagctacga gatcgggcgg ccgttcttgg agaaggccgg ggtggcgcac 480aaggtggact tccgcgaggg gaaggggctg gagaagctgg acgagctgct cgccgaggag 540gcggcggcgg ggcgcgaggc ggcgttcgac ttcgcgttcg tggacgcgga caagcccaac 600tacgtcaagt accacgagca gctgctgcag ctggtgcgcg tcggcgggca catcgtgtac 660gacaacacgc tgtgggccgg cacggtggcg ctgccgccgg acacgccgct gtcggacctg 720gaccggaggt tctccgtcgc catcagggac ctcaactcca ggctcgccgc cgacccgcgc 780atcgacgtct gccagctcgc catcgccgac ggcatcacca tctgccgccg cctcgtgtga 840ggtcgagacc gagaccttac cggccgatcc atccatcgct ctcgcgtgat taattaacgt 900gtgttgctgt actcttctac tgctacaact atactattac ttccttaatt gccgcttaaa 960ttttcctata cgtgtttcaa tcaatgagat tattatattc ttcgagcatg agagagacgg 1020agttgtaggg acatttgatg atggttgtta ctgtactaca tgttgataag tgcaacatct 1080ctttccatgg ttgctactct actcaccgtg tcatgttggt tgcggatttt gatctcatct 1140gcaagatgga ctactggggc ccaaaatgga acagactggt ccctcgatcc tgcaggagct 1200tgcacctgtt gcaagggcct ttttaactgg ctaactaggt gggtaagtag gg 125230671DNAOryza sativamisc_featureTC89670 (PRO0091) 30gcnggcttcg gcangagttc aaacattata gttgaagcat agtagtagaa tcctacaaaa 60atgaagatca ttttcgtatt tgctctcctt gctattgttg catgcaacgc ttctgcacgg 120tttgatgctc ttagtcaaag ttatagacaa tatcaactac aatcgcatct cctgctacag 180caacaagtgc tcagcccatg cagtgagttc gtaaggcaac agcatagcat agtggcaacc 240cccttctggc aaccagctac gtttcaattg ataaacaacc aagtcatgca gcaacagtgt 300tgccaacagc tcaggctggt agcgcaacaa tctcactacc aggccattag tagcgttcag 360gcgattgtgc agcaactaca gctgcagcag gtcggtgttg tctactttga tcagactcaa 420gctcaagctc aagctttgct ggccttaaac ttgccatcca tatgtggtat ctatcctaac 480tactacattg ctccgaggag cattcccacc gttggtggtg tctggtactg aattgtaata 540gtataatggt tcaaatgtta aaaataaagt catgcatcat catgcgtgac agttgaaact 600tgatgtcata taaatctaaa taaaatcacc tatttaaata gcattcatgt atgagttcca 660ttatcatagc t 67131436DNAOryza sativamisc_featureTC89883 (PRO0095) 31cctcgagggt cgacccacgc gtccgctctc ctctcttctc tcgccctcac cgctcgccga 60ggttgccgtc tccttgtctc ctccgctcct tgcgccgccg ccgcgacgag tcgcggggag 120gggcggcgat ctccatctcc atctgaggcg aggagagcag gggaggtgag gggatcctgg 180tgaggtttgt gattactgga caatagaaat atttacacaa tatggctggc ggctctgctg 240atgcagtgac caaggagatg gaggcgctac tcgttggaca aaatccaaat gcggttagtg 300gagaaacatg cgagacctca tcaaaagaag gcaaagttgc agatagcaat ggatctcatt 360cttcaccacc agaagatgat gatgatgaag cgcaagggga tggtccatct caagattgga 420ggatccagaa gctttc 43632860DNAOryza sativamisc_featureTC90434 (PRO0111) 32nagggctaan attaccggag tatttttgca aagggagtaa tcaaagttcc aatacgaaat 60cgcggtcgta gtagtacaat acaaagacga gttcacggag cgcgtaaact aataaggaaa 120aattaaacgt cgcggagaaa taatagccga actggatgaa gatgagcagc actgcctctt 180gcctagccta gcccatcatg gcgaggccga cggccccgac cagcaggccc atcaccgaac 240gggcctcgct gccgctggcc ccgccggtgc tgcccgtcga cttcgtcgtc gtcgtcgtcg 300gcgtcgtggt cgcgtccggc gtcgacgagg gcgtgtccat gccggggtcc gatgacggcg 360tggcgggcgt cgcggtggac ggcggggacg acgacgccgt cggggtgggg gtggtgccgg 420ccgccgcgga gaccgtgacg gcgagcttca tgccgccgga gcagtggccg ctggtgccgc 480agatgaagta gcgggtgccg ggcttggtga gcgcgatctt ggtgttctgg tcgctgtagg 540actggatcga gttgctggcg gacacgcgct gtagtcagcc gagctcacct ccgccaccgt 600gtgcatcatg ctgtactgga acacgagcga gtcaccaacg ctgaaggttt tgctcttcgc 660ccaggtatcg tagtccacgc cactgctcca gccggatgtg tcgccgacgg tgtagtccac 720ggcgaaagcc ggcgcaacgg cggcgaggag tagcaccacc agacctgcag ctgcaagtcc 780atgtactcca gccatgatgg cagagttaat tagcaaacgc gaactgatta gagccgtact 840agtactggtg gccctcgtgc 860331167DNAOryza sativamisc_featureTC83072 (PRO0116) 33aggaaaagaa gaaaaaagat cctgtgaacc ctacgaaact accgaagcga acggaaggca 60ggaatcggcg gcggcggcgg cggcggcggt ggggagaagc catggagcgg ctgcagcgga 120tcttcggcgc ctccggcatg gggcagccgc cgtcggactc gccgctgctc gactcctccg 180agcaggtcta catctcctcc ctcgccctcc tcaagatgct caagcacggg agggccggcg 240tgccgatgga ggtgatgggg ctgatgctgg gggagttcgt cgacgactac acggtcaggg 300tggtcgacgt cttcgccatg ccgcagagcg ggaccggggt cagcgtcgag gccgtcgacc 360atgtcttcca gaccaacatg ctcgacatgc tcaagcagac cgggaggcca gaaatggtgg 420taggttggta ccattcccat cctggatttg gttgctggct ttcaggagtt gacatcaata 480ctcaacagag ttttgaagct ttaaacccca gggcagttgc cgtcgtgata gatcccatcc 540aaagtgtcaa ggggaaagtt gtcattgatg catttcgcct tattaaccct cagaccatga 600tgcttggtca ggagccacga cagacaacat caaatgttgg gcacctaaat aagccatcta 660ttcaggctct tattcatggg ctgaacaggc actactattc aattgcaatc aattaccgga 720aaaatgagct tgaggaaaag atgttactga acttgcacaa aaagaaatgg accgatggat 780tgattctgaa gaggtttgac actcattcaa agaccaatga gcagactgtt caggaaatgc 840tgaaccttgc tatcaagtac aacaaggcgg tgcaagagga ggatgagctg ccgcctgaga 900aattagcgat agcaaatgtg ggacggcaag atgctaagaa gcacttggaa gagcatgtct 960ccaatttgat gtcatcaaac atagttcaga cgctaggaac catgctcgat acagttgtat 1020tttagatcac tactgctgtt atcccaacac tgtacccaga gctcgtttat tttttatttt 1080tttatgttta tcgaagccta ccataattca gtgaacttaa cgccagttac atttgggtta 1140tgaaagctta ccacttgaca acttcat 116734871DNAOryza sativamisc_featureTC90038 (PRO0117) 34cctagctcct cccgccgccg ccgccgccgc cgccgccgcc tctccactcg agagacccag 60ccgccgccgc cgccgccgcc gccatgtcgc tgatcgccgg ggaggacttc cagcacatcc 120tgcgtctgct gaacaccaac gtcgatggga agcagaagat catgttcgcg ctcacctcca 180tcaagggtgt
cggccgcagg ttctccaaca tcgcctgcaa gaaggccgac atcgacatga 240acaagagggc cggtgagctt acgccggagg agctggagcg gctgatgacc gtggtggcga 300acccgcggca gttcaaggtg cccgactggt tcctcaacag gaagaaggac tacaaggacg 360ggaggttctc ccaggttgtc tccaacgcgc tcgacatgaa gctcagggat gatcttgaga 420ggctcaagaa gatcaggaac caccgtggtc tgaggcacta ctggggcctc cgtgtgcgtg 480ggcagcacac caagacaacc ggaaggaggg gtaagactgt cggtgtgtcc aagaagcgat 540aagcctaaga accacccgag acttgatgaa gcgtttcgtt gggtgatgtt ttgccctagg 600ataatatttt gcagctatgg aaccttgtcg taatgtatct tgaagagtgt ctttgggaac 660taagagtaat ttacttttct tgaaactatt gcagtattga ctccttgttt attgcttttc 720tccactttct tctacccact taaaactatt gcagtatcga ctccttgttt attgctattc 780tccactggct tctgccttaa ttttggatgt tgcatgcgct gtgtatctgg ttcatgtgat 840gtacccatgg cagctttgat gcattgggat t 871351245DNAOryza sativamisc_featureTC82936 (PRO0122) 35acgcggccaa aacgtaccct tgtgactaca cccgcttcgc ttcctcccct ctctaagccg 60gggaagctaa gccatggcgt ccgtcaccgc ccgcaccccg gtcgcagccc tccgctcgtc 120ggcgtcgctc aagtctacct tcctagggca atcctccacc cgcctcgccc gcgcaccgac 180tacgaggcgt aatgttcggg cggaggccaa gggagagtgg ctccccggcc tcccttctcc 240cacctacctc aacggcagct tgccaggcga taacgggttc gacccgttgg gtctggcgga 300ggacccggag aacctgcggt ggttcgtgca ggcggagtgg tgaacgggcg gtgggcgatg 360ctgggggtgg ccgggatgct gctgcctgag gtgctgacga agatcgggtt gatcgacgcg 420ccgcagtggt acgacgccgg caaggccacc tacttcgcgt cgtcgtcgac gctgttcgtc 480atcgagttca tcctgttcca ctacgtggag atccggcggt ggcaggacat caagaaccct 540ggctgcgtca accaggaccc catcttcaag agctacagcc tcccgccgca cgagtgcggc 600taccccggca gcgtcttcaa ccccctcaac ttcgagccca ccctcgaggc caaggagaag 660gagctcgcca acgggaggct ggcgatgctg gcgttcttgg ggttcctggt gcagcacaac 720gtgacgcaga aggggccctt cgacaacctg ctgcagcacc tgtctgaccc gtggcacaac 780accatcatcc agacgctgtc aggctgagcg tgtgatcgat ttcatcaggg ccagggcatc 840tcaaggagct tgatgagttc aggctggtga aaccgatgat tgggcgatgg aagatgttct 900cttcttgttt cttctttttt tttttgtgga gtatgcatgt ataagatgtt aatgaattgg 960ggggaggaga gagagagaga tggatgtgat gagattcaga cttactgtgt gtgttgtggt 1020aattgtttcc tgcatgcatg gatctggatg catgggtgag ggggtgagtt gagtggtgaa 1080tttctgatgt acagtactac agggggataa actatctcat ggtagcagca gtgttctagc 1140tatctcatgg tctcgatctt aattatggtg gataaactac gcttaattgc ttgtcaagtg 1200cttcatttgc gcattgattc agtattgcgt atcgattcaa agacc 1245361416DNAOryza sativamisc_featureTC89839 (PRO0123) 36cccacgcgtc cgcccacgcg tccgggacac cagaaacata gtacacttga gctcactcca 60aactcaaaca ctcacaccaa tggctctcca agttcaggcc gcactcctgc cctctgctct 120ctctgtcccc aagaagggta acttgagcgc ggtggtgaag gagccggggt tccttagcgt 180gagcagaagg ccaagaagcc gtcgctggtg gtgagggcgg tggcgacgcg gcgggccggt 240ggcgagcccc ggcgcgggca cgtcgaaggc ggacgggaag aagacgctgc ggcagggggt 300ggtggtgatc accggcgcgt cgtcggggct cgggctcgcg gcggcgaagg cgcttggcgg 360agacggggaa gtggcacgtg gtgatggcgt tccgcgactt tcctgaaggc ggcgacggcg 420gcgaaggcgg cggggatggc ggcggggagc tacaccgtca tgcacctgga cctcgcctcc 480ctcgacagcg tccgccagtt cgtggacaac ttccggcgct ccggcatgcc gctcgacgcg 540ctggtgtgca acgccgcaca tctaccggcc gacggcgcgg caaccgacgt tcaacgccga 600cgggtacgag atgagcgtcg gggtgaacca cctgggccac ttcctcctcg cccgcctcat 660gctcgacgac ctcaagaaat ccgactaccc gtcgcggcgg ctcatcatcc tcggctccat 720caccggcaac accaacacct tcgccggcaa cgtccctccc aaggccgggc taggcgacct 780ccgggggctc gccggcgggc tccgcgggca gaacgggtcg gcgatgatcg acggcgcgga 840gagcttcgac ggcgccaagg cgtacaagga cagcaagatc tgtaacatgc tgacgatgca 900ggagttccac cggagattcc acgaggagac cgggatcacg ttcgcgtcgc tgtacccggg 960gtgcatcgcg acgacgggct tgttccgcga gcacatcccg ctgttccggc tgctgttccc 1020gccgttccag cggttcgtga cgaaggggtt cgtgtcggag gcggagtccg ggaagcggct 1080ggcgcaggtg gtgggcgacc cgagcctgac caagtccggc gtgtactgga gctggaacaa 1140ggactcggcg tcgttcgaga accagctctc gcaggaggcc agcgacccgg agaaggccag 1200gaagctctgg gacctcagcg agaagctcgt cggcctcgtc tgagtttatt atttacccat 1260tcgtttcaac tgttaatttc ttcggggttt agggggtttc agctttcagt gagagaggcc 1320tgtcaagtga tgtacaatta gtaatttttt tttacccgac aaatcatgca ataaaaccac 1380aggcttacat tatcgatttg tccacctaaa ttaagt 1416371149DNAOryza sativamisc_featureTC85888 (PRO0133) 37cttctacttc tatcatacca aacaaactag cttaatttgc attgcatcac attgccggcc 60gccatgagag ctctcgctct cgcggtggtg gccatggcgg tggtggccgt gcgcggcgag 120cagtgcggca gccaggccgg cggcgcgctc tgccccaact gcctctgctg cagccagtac 180ggctggtgcg gctccacctc cgattactgc ggcgccggct gccagagcca gtgctccggc 240ggctgcggcg gcggcccgac cccgccctcc agcggtggcg gcagcggcgt cgcctccatc 300atatcgccct cgctcttcga ccagatgctg ctccaccgca acgaccaggc gtgcgccgct 360aagggcttct acacctacga cgccttcgtc gccgccgcca acgcctaccc ggacttcgcc 420accacccgcg acgccgacac ctgcaagcgc gaggtcgccg ccttcctggc gcagacgtcc 480cacgagacca ccggcggctg gcccacggcg cccgacggcc cctactcctg gggctactgc 540ttcaaggagg agaacaacgg caacgccccc acatactgcg agcccaagcc ggagtggccg 600tgcgccgccg cgaagaagta ctacggccgg ggacccatcc agatcaccta caactacaac 660tacggccgcg gggcaggcat cggctccgac ctgctcaaca acccggacct ggtggcgtcg 720gacgccagtc tccttcaaga cggcgttctg gttctggatg acgccgcagt cgcccaagcc 780gtcgtgccac gcggtgatca ccggccagtg gacgccgtcc gccgacgacc aggcggcggg 840gcgcgttccg ggctacggcg agatcaccaa catcatcaac ggcggtgtgg agtgcgggca 900cggcgcggac gacaaggtgg ccgaccggat cgggttctac aagcgctact gcgacatgct 960gggcgtcagc tatggcgata acctggattg ctacaaccag aggccctacc cgccttccta 1020gttgatattt gatccgagca gacgaataaa atacaatgca cacgagattg tgagactcga 1080gaaaacatat actacctctg aattttaata catatctcta aaacaaaaaa aaaaaaaaaa 1140aaaatatac 114938981DNAOryza sativamisc_featureTC84300 (PRO0151) 38aagaggcaag agcatccgta ttaaccagcc ttttgagact tgagagtgtg tgtgactcga 60tccagcgtag tttcagttcg tgtgttggtg agtgattcca gccaagtttg cgatggcttc 120tcagcaggaa cgggctagct accacgccgg cgagaccaag gcccgcgccg aggagaagac 180ggggcgcatg atgggcacgg cgcaggagaa ggcgcgggag gccaaggaca cggcgtccga 240cgccgcgggg cgcgcgatgg gcaggggaca cggcgccaag gaggcgacca aggagaaggc 300gtacgagacc aaggacgcga ccaaggagaa ggcgtacgag gcaaaggacg cggcctccga 360cgccaccggc cgcgccatgg acaagggccg cggcgccgcg ggcgccacga gggacaaggc 420gtacgatgcc aaggacaggg cggctgacac ggcgcagtcc gccgccgacc gcgcccgcga 480cggcgccggg cagaccggga gctacattgg acagaccgcc gaggccgcca agcagaaagc 540ggccggcgcc gcgcagtacg ccaaggagac cgcgatcgcc ggcaaggaca agaccggcgc 600cgtgctccag caggcagggg agcaggtgaa gagcgtggcg gtgggggcga aggacgcggt 660gatgtacacg ctcgggatgt caggcgataa caagaacaac gccgctgccg gcaaggacac 720cagcacctac aagcctggaa ctgggagtga ctaccagtaa tacggtagaa gaagcatgtg 780tcgtctttgg cactgatgcc aaagtgtacg tgttgtatcc tcttttttaa gtttcagctc 840gacttcgacg tgttcggtgt cacactttgg tttttcagtt gtgctcaact gttcatgttt 900ctggttccat ggagggccag tgtggaggtc aatgtttaag ctttcgtttt aaaatctgat 960aataaagttg gttaagacct g 981391203DNAOryza sativamisc_featureTC89687 (PRO0169) 39tactcctctc tctcacctcc accatctagc tcactcacac agtctccact cacacgcatt 60gcagaggaga ggcgacaatg gaggggaagg aggaggacgt gcggctgggg gcgaacaggt 120actcggagag gcagccgata gggacggcgg cgcagggcgc gggggacgac aaggactaca 180aggagccgcc gccgggccgc tgttcgagcc aggggagctc aagtcgtggt ctttctaccg 240ggccgggatc gccgagttcg tcgccacctt cctcttcctc tacatcacca tcctcaccgt 300catgggggtc tccaagtcct cctccaagtg cgccaccgtc ggcatccagg gcatcgcctg 360gtccttcgga ggcatgatct tcgcgctcgt ctactgcacc gccggcatct ccggaggaca 420catcaaccca gcagttactt ttgggctgtt cttggccagg aagctgtccc tgacccgggc 480catcttctac atagtgatgc aatgcctagg ggccatctgc ggagctggag ttgtgaaggg 540cttccagcag ggtctgtaca tgggcaatgg cggtggtgcc aatgtagttg ccagtggcta 600caccaagggt gacggtcttg gtgctgagat tgttggcacc ttcatcctgg tctacaccgt 660cttctcagcc actgatgcca agaggaatgc cagggactca catgttccta tccttgcccc 720actgccaatt ggttttgcgg tgttcctggt ccacctggcc accatcccca tcaccggtac 780tggcatcaac ccagccagga gccttggcgc tgccatcatc tacaacaagg accatgcctg 840gaatgaccat tggatcttct gggttggtcc cttcgttggc gctgccctgg ctgccatcta 900ccaccaggtg atcatcaggg cgatcccatt caagagcagg tcttaagccc cgcgccgccg 960ctgcgcagcc gacgacatgc aacgcaatcg tgatgtcctg tttcccgcgc gctactgctg 1020cgcatctgtc gattccctct atctctagtc cccaagatgt ttttcctatc tgaaccctga 1080acaactcaat cgtgtaatcc agtactcagt cactgtatgt ttttatgtga tggagatctt 1140aattcttaag ttatcatctc tgttgctgga aatccggttt cctcttcgtg catgaaccgc 1200gcc 120340964DNAOryza sativamisc_featureTC89846 (PRO0170) 40cccacggttc cgcccacggt ccgcccacgg tccgcttctc ttctctggtg gtgtgggtgt 60gtccctgtct cccctctcct tcctcctctc ctttcccctc ctctcttccc ccctctcaca 120agagagagag cgccagactc tccccaggtg aggattcagc catgaagggg gccaaatcca 180agggcgccgc caagcccgac gccaagttgg ctgtgaagag taagggcgcg gagaagcccg 240ccgccaaggg caggaagggg aaggccggca aggaccccaa caagcccaag agggctccct 300ccgctttctt cgtttttatg gaggagttcc gtaaggagtt caaggagaag aaccccaaga 360ataaatctgt cgctgctgta ggaaaagcag ccggtgatag gtggaaatcc ctgaccgaag 420cggacaaggc tccctatgta gccaaggcca acaagctcaa ggccgagtac aacaaggcca 480ttgctgccta caacaagggc gagagcactg ccaagaaggc acccgccaag gaggaagagg 540aggacgacga ggaggaatct gacaagtcca agtccgaggt caatgatgag gatgacgacg 600agggcagcga agaggatgaa gacgatgacg agtgagcctt ccagtggaca agatgggagc 660agcaagacgc taagggcggc gggcgtccta aggagcctat ccatcatcat catcgtctac 720tagaattatt cagtttcact tcacatcgtg atgttttact ttttctctcg tcctataacg 780gatagcgctc cttgttggcg ccactggtgg gtgttgtggt gcagccaatg tcttgtctcc 840accgtcaatg atccgcttgt acctagatta ctctttccat tgtcatcggc taacattgtg 900ataatatcag tttgcgtatg ttagattaaa ttgtttctaa ttccgtcgtt ttcttcttcc 960ttgc 964411542DNAOryza sativamisc_featureTC82935 (PRO0171) 41cacacctcac acctcaccac catcacctcc tcctcctcct cctcttcctc cgcgcgcgcg 60agatccaggg agagggagag ggagagatca tggcggggac ggtgacggtg ccgtcggcgt 120cggtgccgtc gacgccgctg ctcaaggacg agctggacat cgtgatcccg acgatccgca 180acctggactt cctggagatg tggcggccct tcttccagcc ctaccacctc atcatcgtgc 240aggacggcga cccgaccaag accatccgcg tccccgaggg cttcgactac gagctctaca 300accgcaacga catcaaccgg atcctcggcc ccaaggcctc ctgcatctcc ttcaaggact 360ccgcatgccg ctgcttcggc tacatggtct ccaagaagaa gtacgtcttc accatcgacg 420acgactgctt cgttgccaag gacccatctg gcaaggacat caatgctctt gagcagcaca 480tcaagaacct cctcagcccg tccaccccgt tcttcttcaa caccttgtat gatccctacc 540gcgaaggcgc tgactttgtc cgtggttacc ccttcagcct cagggaggga gccaagactg 600ctgtctctca cggcctgtgg cttaacatcc ctgactatga tgctcctact cagatggtca 660agcctcgtga gaggaactcc aggtatgttg atgctgtcat gactgtgccc aagggaacct 720tgttccccat gtgtggcatg aaccttgctt ttgaccgtga tctcatcggt cctgcaatgt 780actttggtct catgggtgat ggccagccta ttggtcgcta cgacgacatg tgggctggat 840ggtgcatgaa ggtcatctgt gaccacctga gcctgggagt gaagactgga ctgccgtaca 900tctggcacag caaggctagc aaccccttcg tgaacttgaa gaaggaatac aagggcatct 960tctggcagga ggacatcatc cccttcttcc agaacgccac catccccaag gagtgcgaca 1020ccgtccagaa gtgctacctc tccctcgccg agcaggtcag ggagaagctc ggcaagatcg 1080acccctactt cgtcaagctt gccgatgcca tggtcacctg gatcgaggcc tgggatgagc 1140tgaacccctc gactgctgct gtcgagaacg gcaaggccaa gtagattgat cctgggagct 1200tgtgtgtcgc aggatggaaa gtacccttta agtgaaagtg ttgctgtggc ctaggccccc 1260tagatatagc tctttttgag atgaagggag agattactta agcaacttta taattctttg 1320ttgttatgct ggttcttttg tagctggaaa aggatttgtt atcatcgttt acataattca 1380agacaataat aattttatca tgtaattttg atagtcgtgc tttggttgct aaatggtgtt 1440attgtattta ataacctttg caaatcacta tacctgttgg ttgttctgag aattgtatgc 1500actaccatat tatatttcta aatcatttcg taggcattat gg 1542421432DNAOryza sativamisc_featureTC82977 (PRO0173) 42aaaagagcag cgtcgcctct cctcctccct aacccctacg cttccagaac cttctcgaag 60ctcccgctcc cccccccctt ccgctccaat ggcgaaggaa ccgatgcgcg tgctcgtcac 120cggcgccgca ggacaaattg gatatgctct tgtccccatg attgctaggg gtgtgatgtt 180gggtgctgac cagcctgtta ttctacacat gcttgacatt ccaccagcta ctgaatctct 240taatggcctt aagatggagc tggttgatgc tgcatttcct cttttgaagg gaattgtcgc 300aacaactgat gttgtggagg cctgcactgg tgtgaatgtt gcggttatgg ttggtgggtt 360ccccaggaag gagggaatgg aaaggaagga tgttatgtca aaaaatgtct ccatctacaa 420atcccaagct tctgctcttg aggctcatgc agcccctaac tgcaaggttc tggtagttgc 480caatccagca aacaccaacg ctctcatctt aaaagaattc gctccatcca tccctgagaa 540gaacattact tgcctcaccc gtcttgacca caacagggca cttggccaga tctctgaaaa 600acttaatgtc caagttactg atgtgaagaa tgcgatcatc tggggcaacc actcatccac 660ccagtaccct gatgttaacc acgccactgt gaagactccc agtggagaga agcctgtcag 720ggaactcgtt gctgatgatg agtggttaaa tacggaattc atctctaccg tccagcagcg 780tggtgccgcc atcatcaagg cgaggaagca atccagtgcc ctatctgctg ccagctctgc 840atgcgatcac attcgtgact gggttcttgg cactcctgag ggaacatttg tctccatggg 900tgtgtactct gatggttcgt atggtgtgcc tgctggtctg atctactcgt tcccagtaac 960atgcagtggt ggcgaatgga cgattgttca gggtctcccg atcgacgagt tctcaaggaa 1020gaagatggac gcgactgccc aggagctgtc ggaggagaag acgctcgctt actcatgcct 1080caactaaaac taagcaatac ccagagggac agatagtgag cgattgcccg ctcccgtgtt 1140tttgaataaa agagactttt aagttccatc acatagaaac tgtttatctc agaccgctgc 1200acatcgcgag atgtggagcg cagatgccgt tgctggtttt actccagtgt gtattgaggc 1260tttgtactag ctcccttttt tttgcctggt gattcgcagg acatttgctg aaaacattga 1320acccatttga catctgatgg aatcatggac cagtagcaag tacatttttg cgaaagcata 1380atctgcatcg ggcttgggct ggtggttgaa ctttctgcca catggcccnt gg 143243659DNAOryza sativamisc_featureTC83646 (PRO0175) 43gctaagtgag ctagccactg atcagaagaa cacctcgatc tctgagagtg ttttttcagc 60tttagcttaa gcaggatgga gcaccagggg cagcacggcc acgtgaccag ccgcgtcgac 120gagtacggca acccggtcgg caccggcgcc ggacacggcc agatgggcac cgccggcatg 180gggacgcacg gcaccgccgg caccggcggc ggccagttcc agccgatgag ggaggagcac 240aagaccggcg gcgtcctgca acgctccggc agctccagct caagctcgtc tgaggatgat 300ggaatgggag ggaggaggaa gaaggggatc aaggagaaga tcaaggagaa gctccccggc 360ggcaacaagg gcgagcagca gcatgccatg ggcggcaccg gcaccggcac cggcaccggc 420accggaaccg gcggcgccta cgggcagcag ggccacggca ccgggatgac caccggcacc 480accggcgcac acggcaccac caccaccgac accggcgaga agaagggcat catggacaag 540atcaaggaga agctgcccgg ccagcactga gctcgacaca ccaccacacc atgtgtctgc 600gcccccggcg accgccgcca cgtcaccttc ctgaataata agatgagcta accgagcgc 659441310DNAOryza sativamisc_featureTC90619 (PRO0177) 44ggaccagcga gcaaccagcc ccccgccccc aatggcggca gagcagcttt gcccaccgct 60gccgcttttg cccacctctc ctccgattaa tcccctcccc tcctcttcct cccacttctc 120cgcctcctct tcctcccctc gccgacccta cctactcgcg ccgccgccgt cgcattgggc 180ggcaaacgga gggggggtta accctgatgg agcagtacga gaaggaggag aagattgggg 240agggcacgta cggggtggtg tacagggcgc gggacaaggt caccaacgag acgatcgcgc 300tcaagaagat ccggcttgag caggaggatg agggcgtccc ctccaccgca atccgcgaga 360tctcgctcct caaggagatg catcacggca acatcgtcag gttacacgat gttatccaca 420gtgagaagcg catatatctt gtctttgagt atctggatct ggacctaaag aagttcatgg 480actcttgtcc agagtttgcg aaaaacccca ctttaattaa gtcatatctc tatcagatac 540tccgcggcgt tgcttactgt cattctcata gagttcttca tcgagatttg aaacctcaga 600atttattgat agatcggcgt actaatgcac tgaagcttgc agactttggt ttagccaggg 660catttggaat tcctgtccgc acgtttactc acgaggttgt aaccttgtgg tatagagctc 720cagagatcct tcttggatca aggcagtatt ctacaccagt tgatatgtgg tcagttggtt 780gtatctttgc agaaatggtg aaccagaaac cactgttccc tggtgattct gagattgatg 840aattatttaa gatattcagg gtactaggaa ctccaaatga acaaagttgg ccaggagtta 900gctcattacc tgactacaag tctgctttcc ccaagtggca agcacaggat cttgcaacta 960ttgtccctac tcttgaccct gctggtttgg accttctctc taaaatgctt cggtacgagc 1020caaacaaaag gatcacagct agacaggctc ttgagcatga atacttcaag gaccttgaga 1080tggtacaatg accctgctat ggctttacat tggattggca tatgtatggg ctgggctcct 1140catttcattc cttctgtgaa cgctgtgccc ttcgtttggg catttttgtc attcagctgg 1200atatttcaaa tcttgtgtgt ttgatatgta ttcaggaacg ctaaatagat caccgtcttg 1260gtctctattt gttcagagta aatatcttcc aatgctgcct ttcagtttcc 13104555DNAArtificial sequenceprm3780 45ggggacaagt ttgtacaaaa aagcaggctt cgacgctact caagtggtgg gaggc 554655DNAArtificial sequenceprm2768 46ggggacaagt ttgtacaaaa aagcaggctc ccgatttagt agaccacatt ttggc 554754DNAArtificial sequenceprm2420 47ggggacaagt ttgtacaaaa aagcaggcta tgccatcgag tggtgtgccg atac 544854DNAArtificial sequenceprm2853 48ggggacaagt ttgtacaaaa aagcaggctt ctcttctgaa gctgaagccc tgcg 544953DNAArtificial sequenceprm2426 49ggggacaagt ttgtacaaaa aagcaggcta aaaccaccga gggacctgat ctg 535055DNAArtificial sequenceprm2855 50ggggacaagt ttgtacaaaa aagcaggctc ctagctatat gcagaggttg acagg 555153DNAArtificial sequenceprm3025 51ggggacaagt ttgtacaaaa aagcaggcta tggtgccatg tcaataagac atc 535256DNAArtificial sequenceprm3029 52ggggacaagt ttgtacaaaa aagcaggctg tttttctatg aaccggtcat taaacc 565355DNAArtificial sequenceprm3061 53ggggacaagt ttgtacaaaa aagcaggctc ctgatggatg atgaatcact gatcg 555457DNAArtificial sequenceprm3031 54ggggacaagt ttgtacaaaa aagcaggctt cgttaagttt gatgatttct gatgacc 575553DNAArtificial sequenceprm3051 55ggggaccact ttgtacaaga aagctgggtg ccgccgctcg ctcgcttcgt tcg 535658DNAArtificial sequenceprm3592 56ggggacaagt ttgtacaaaa aagcaggctc gtgttcatgt tcgcatttag gattggac 585755DNAArtificial sequenceprm5131 57ggggacaagt ttgtacaaaa aagcaggctc agatgccaca gtatggtgta ccacc 555856DNAArtificial sequenceprm3782 58ggggacaagt ttgtacaaaa aagcaggctt tgcagttgtg accaagtaag ctgagc 565954DNAArtificial sequenceprm2844 59ggggacaagt ttgtacaaaa aagcaggctt ttggcgcggg gcagaagagt ggac 546057DNAArtificial sequenceprm2973 60ggggacaagt ttgtacaaaa aagcaggctg cttgagtcat agggagaaaa caaatcg 576153DNAArtificial sequenceprm3770 61ggggacaagt ttgtacaaaa aagcaggctc gtcctccttt tgtaacggct cgc 536256DNAArtificial sequenceprm3772 62ggggacaagt ttgtacaaaa aagcaggctc
atgcggctaa tgtagatgct cactgc 566353DNAArtificial sequenceprm3774 63ggggacaagt ttgtacaaaa aagcaggctt agtaccattc ttccctcgtg agc 536453DNAArtificial sequencepm3776 64ggggacaagt ttgtacaaaa aagcaggctg tttggttggt gaccgcaatt tgc 536555DNAArtificial sequenceprm3800 65ggggacaagt ttgtacaaaa aagcaggctg tcaccaccgt catgtacgag gctgc 556655DNAArtificial sequenceprm5135 66ggggacaagt ttgtacaaaa aagcaggctc agacacctag aatatagaca ttccc 556755DNAArtificial sequenceprm3781 67ggggaccact ttgtacaaga aagctgggtg atcacaagcg cagctaatca ctagc 556857DNAArtificial sequenceprm2769 68ggggaccact ttgtacaaga aagctgggtc gtgtagaaaa tcttaacccg aaaatcg 576955DNAArtificial sequenceprm2421 69ggggaccact ttgtacaaga aagctgggtg gtgaggtgcc ggggaagcga cgttg 557054DNAArtificial sequenceprm2854 70ggggaccact ttgtacaaga aagctgggtt tcttctttcc cttggaacta accg 547154DNAArtificial sequenceprm2427 71ggggaccact ttgtacaaga aagctgggtt gtcgctttta tttggcttgg tgtg 547256DNAArtificial sequenceprm2856 72ggggaccact ttgtacaaga aagctgggtc tctagctcga tctctcttgc aaaagc 567349DNAArtificial sequenceprm3026 73ggggaccact ttgtacaaga aagctgggtg gcgatgagat cttcctccg 497459DNAArtificial sequenceprm3030 74ggggaccact ttgtacaaga aagctgggtt tttgtaggat tctactacta tgcttcaac 597562DNAArtificial sequenceprm3062 75ggggaccact ttgtacaaga aagctgggta ttgtgtaaat atttctattg tccagtaatc 60ac 627654DNAArtificial sequenceprm3032 76ggggaccact ttgtacaaga aagctgggtg atggcagagt taattagcaa acgc 547750DNAArtificial sequenceprm3052 77ggggacaagt ttgtacaaaa aagcaggctc taagggcagc agccattggg 507860DNAArtificial sequenceprm3049 78ggggaccact ttgtacaaga aagctgggtg gcggcggcgg cggcggcggc ggctgggtct 607954DNAArtificial sequenceprm2195 79ggggaccact ttgtacaaga aagctgggtc ggcttagaga ggggaggaag cgaa 548058DNAArtificial sequenceprm2197 80ggggaccact ttgtacaaga aagctgggtt ggtgtgagtg tttgagtttg gagtgagc 588157DNAArtificial sequenceprm2845 81ggggaccact ttgtacaaga aagctgggtc ggcaatgtga tgcaatgcaa attaagc 578255DNAArtificial sequenceprm2974 82ggggaccact ttgtacaaga aagctgggtc gcaaacttgg ctggaatcac tcacc 558354DNAArtificial sequenceprm3771 83ggggaccact ttgtacaaga aagctgggtt gtcgcctctc ctctgcaatg cgtg 548452DNAArtificial sequenceprm3773 84ggggaccact ttgtacaaga aagctgggtg gctgaatcct gcgagaaggg cg 528554DNAArtificial sequenceprm3775 85ggggaccact ttgtacaaga aagctgggtg atctctccct ctccctctcc ctgg 548652DNAArtificial sequenceprm3777 86ggggaccact ttgtacaaga aagctgggtt ggagcggaag ggggggggga gc 528757DNAArtificial sequenceprm3801 87ggggaccact ttgtacaaga aagctgggtc actctcagag atcgaggtgt tcttctg 578852DNAArtificial sequenceprm5136 88ggggaccact ttgtacaaga aagctgggtc gcccgcagct cgcccccgtc cg 52
Patent applications by Willem Broekaert, Dilbeek BE
Patent applications by Yves Hatzfeld, Lille FR